Datasets:

OTAR3088
/

Variants_IOB

words	labels
The	O
Bacteroidetes	O
are	O
dominant	O
bacteria	O
in	O
the	O
human	O
gut	O
that	O
are	O
responsible	O
for	O
the	O
digestion	O
of	O
the	O
complex	O
polysaccharides	O
that	O
constitute	O
“	O
dietary	O
fiber	O
.”	O
Although	O
this	O
symbiotic	O
relationship	O
has	O
been	O
appreciated	O
for	O
decades	O
,	O
little	O
is	O
currently	O
known	O
about	O
how	O
Bacteroidetes	O
seek	O
out	O
and	O
bind	O
plant	O
cell	O
wall	O
polysaccharides	O
as	O
a	O
necessary	O
first	O
step	O
in	O
their	O
metabolism	O
.	O
	O
Here	O
,	O
we	O
provide	O
the	O
first	O
biochemical	O
,	O
crystallographic	O
,	O
and	O
genetic	O
insight	O
into	O
how	O
two	O
surface	O
glycan	O
-	O
binding	O
proteins	O
from	O
the	O
complex	O
Bacteroides	O
ovatus	O
xyloglucan	O
utilization	O
locus	O
(	O
XyGUL	O
)	O
enable	O
recognition	O
and	O
uptake	O
of	O
this	O
ubiquitous	O
vegetable	O
polysaccharide	O
.	O
	O
More	O
importantly	O
,	O
this	O
makes	O
diet	O
a	O
tractable	O
way	O
to	O
manipulate	O
the	O
abundance	O
and	O
metabolic	O
output	O
of	O
the	O
microbiota	O
toward	O
improved	O
human	O
health	O
.	O
	O
The	O
archetypal	O
PUL	O
-	O
encoded	O
system	O
is	O
the	O
starch	O
utilization	O
system	O
(	O
Sus	O
)	O
(	O
Fig	O
.	O
1B	O
)	O
of	O
Bacteroides	O
thetaiotaomicron	O
.	O
	O
The	O
location	O
of	O
SGBP	O
-	O
A	O
/	O
B	O
is	O
presented	O
in	O
this	O
work	O
;	O
the	O
location	O
of	O
GH5	O
has	O
been	O
empirically	O
determined	O
,	O
and	O
the	O
enzymes	O
have	O
been	O
placed	O
based	O
upon	O
their	O
predicted	O
cellular	O
location	O
.	O
	O
We	O
recently	O
reported	O
the	O
detailed	O
molecular	O
characterization	O
of	O
a	O
PUL	O
that	O
confers	O
the	O
ability	O
of	O
the	O
human	O
gut	O
commensal	O
B	O
.	O
ovatus	O
ATCC	O
8483	O
to	O
grow	O
on	O
a	O
prominent	O
family	O
of	O
plant	O
cell	O
wall	O
glycans	O
,	O
the	O
xyloglucans	O
(	O
XyG	O
).	O
	O
As	O
the	O
Sus	O
SGBPs	O
remain	O
the	O
only	O
structurally	O
characterized	O
cohort	O
to	O
date	O
,	O
we	O
therefore	O
wondered	O
whether	O
such	O
glycan	O
binding	O
and	O
function	O
are	O
extended	O
to	O
other	O
PUL	O
that	O
target	O
more	O
complex	O
and	O
heterogeneous	O
polysaccharides	O
,	O
such	O
as	O
XyG	O
.	O
	O
These	O
data	O
extend	O
our	O
current	O
understanding	O
of	O
the	O
Sus	O
-	O
like	O
glycan	O
uptake	O
paradigm	O
within	O
the	O
Bacteroidetes	O
and	O
reveals	O
how	O
the	O
complex	O
dietary	O
polysaccharide	O
xyloglucan	O
is	O
recognized	O
at	O
the	O
cell	O
surface	O
.	O
	O
Similarly	O
,	O
SGBP	O
-	O
B	O
also	O
bound	O
to	O
XyG	O
and	O
XyGO2	O
with	O
approximately	O
equal	O
affinities	O
,	O
although	O
in	O
both	O
cases	O
,	O
Ka	O
values	O
were	O
nearly	O
10	O
-	O
fold	O
lower	O
than	O
those	O
for	O
SGBP	O
-	O
A	O
.	O
Also	O
in	O
contrast	O
to	O
SGBP	O
-	O
A	O
,	O
SGBP	O
-	O
B	O
also	O
bound	O
to	O
XyGO1	O
,	O
yet	O
the	O
affinity	O
for	O
this	O
minimal	O
repeating	O
unit	O
was	O
poor	O
,	O
with	O
a	O
Ka	O
value	O
of	O
ca	O
.	O
1	O
order	O
of	O
magnitude	O
lower	O
than	O
for	O
XyG	O
and	O
XyGO2	O
.	O
	O
As	O
anticipated	O
by	O
sequence	O
similarity	O
,	O
the	O
high	O
-	O
resolution	O
tertiary	O
structure	O
of	O
apo	O
-	O
SGBP	O
-	O
A	O
(	O
1	O
.	O
36	O
Å	O
,	O
Rwork	O
=	O
14	O
.	O
7	O
%,	O
Rfree	O
=	O
17	O
.	O
4	O
%,	O
residues	O
28	O
to	O
546	O
)	O
(	O
Table	O
2	O
)	O
displays	O
the	O
canonical	O
“	O
SusD	O
-	O
like	O
”	O
protein	O
fold	O
dominated	O
by	O
four	O
tetratrico	O
-	O
peptide	O
repeat	O
(	O
TPR	O
)	O
motifs	O
that	O
cradle	O
the	O
rest	O
of	O
the	O
structure	O
(	O
Fig	O
.	O
4A	O
).	O
	O
Cocrystallization	O
of	O
SGBP	O
-	O
A	O
with	O
XyGO2	O
generated	O
a	O
substrate	O
complex	O
structure	O
(	O
2	O
.	O
3	O
Å	O
,	O
Rwork	O
=	O
21	O
.	O
8	O
%,	O
Rfree	O
=	O
24	O
.	O
8	O
%,	O
residues	O
36	O
to	O
546	O
)	O
(	O
Fig	O
.	O
4A	O
and	O
B	O
;	O
Table	O
2	O
)	O
that	O
revealed	O
the	O
distinct	O
binding	O
-	O
site	O
architecture	O
of	O
the	O
XyG	O
binding	O
protein	O
.	O
	O
Seven	O
of	O
the	O
eight	O
backbone	O
glucosyl	O
residues	O
of	O
XyGO2	O
could	O
be	O
convincingly	O
modeled	O
in	O
the	O
ligand	O
electron	O
density	O
,	O
and	O
only	O
two	O
α	O
(	O
1	O
→	O
6	O
)-	O
linked	O
xylosyl	O
residues	O
were	O
observed	O
(	O
Fig	O
.	O
4B	O
;	O
cf	O
.	O
	O
The	O
functional	O
importance	O
of	O
this	O
platform	O
is	O
underscored	O
by	O
the	O
observation	O
that	O
the	O
W82A	B-mutant
W283A	B-mutant
W306A	B-mutant
mutant	O
of	O
SGBP	O
-	O
A	O
,	O
designated	O
SGBP	B-mutant
-	I-mutant
A	I-mutant
*,	I-mutant
is	O
completely	O
devoid	O
of	O
XyG	O
affinity	O
(	O
Table	O
3	O
;	O
see	O
Fig	O
.	O
S4	O
in	O
the	O
supplemental	O
material	O
).	O
	O
Protein	O
name	O
Ka	O
ΔG	O
(	O
kcal	O
⋅	O
mol	O
−	O
1	O
)	O
ΔH	O
(	O
kcal	O
⋅	O
mol	O
−	O
1	O
)	O
TΔS	O
(	O
kcal	O
⋅	O
mol	O
−	O
1	O
)	O
Fold	O
changeb	O
M	O
−	O
1	O
SGBP	O
-	O
A	O
(	O
W82A	B-mutant
W283A	B-mutant
W306A	B-mutant
)	O
ND	O
NB	O
NB	O
NB	O
NB	O
SGBP	O
-	O
A	O
(	O
W82A	B-mutant
)	O
c	O
4	O
.	O
9	O
9	O
.	O
1	O
×	O
104	O
−	O
6	O
.	O
8	O
−	O
6	O
.	O
3	O
0	O
.	O
5	O
SGBP	O
-	O
A	O
(	O
W306	O
)	O
ND	O
NB	O
NB	O
NB	O
NB	O
SGBP	O
-	O
B	O
(	O
230	O
–	O
489	O
)	O
0	O
.	O
7	O
(	O
8	O
.	O
6	O
±	O
0	O
.	O
20	O
)	O
×	O
104	O
−	O
6	O
.	O
7	O
−	O
14	O
.	O
9	O
±	O
0	O
.	O
1	O
−	O
8	O
.	O
2	O
SGBP	O
-	O
B	O
(	O
Y363A	B-mutant
)	O
19	O
.	O
7	O
(	O
2	O
.	O
9	O
±	O
0	O
.	O
10	O
)	O
×	O
103	O
−	O
4	O
.	O
7	O
−	O
18	O
.	O
1	O
±	O
0	O
.	O
1	O
−	O
13	O
.	O
3	O
SGBP	O
-	O
B	O
(	O
W364A	B-mutant
)	O
ND	O
Weak	O
Weak	O
Weak	O
Weak	O
SGBP	O
-	O
B	O
(	O
F414A	B-mutant
)	O
3	O
.	O
2	O
(	O
1	O
.	O
80	O
±	O
0	O
.	O
03	O
)	O
×	O
104	O
−	O
5	O
.	O
8	O
−	O
11	O
.	O
4	O
±	O
0	O
.	O
1	O
−	O
5	O
.	O
6	O
	O
Binding	O
thermodynamics	O
are	O
based	O
on	O
the	O
concentration	O
of	O
the	O
binding	O
unit	O
,	O
XyGO2	O
.	O
	O
Domains	O
A	O
,	O
B	O
,	O
and	O
C	O
display	O
similar	O
β	O
-	O
sandwich	O
folds	O
;	O
domains	O
B	O
(	O
residues	O
134	O
to	O
230	O
)	O
and	O
C	O
(	O
residues	O
231	O
to	O
313	O
)	O
can	O
be	O
superimposed	O
onto	O
domain	O
A	O
(	O
residues	O
34	O
to	O
133	O
)	O
with	O
RMSDs	O
of	O
1	O
.	O
1	O
and	O
1	O
.	O
2	O
Å	O
,	O
respectively	O
,	O
for	O
47	O
atom	O
pairs	O
(	O
23	O
%	O
and	O
16	O
%	O
sequence	O
identity	O
,	O
respectively	O
).	O
	O
While	O
there	O
is	O
no	O
substrate	O
-	O
complexed	O
structure	O
of	O
Bacova_04391	O
available	O
,	O
the	O
binding	O
site	O
is	O
predicted	O
to	O
include	O
W241	O
and	O
Y404	O
,	O
which	O
are	O
proximal	O
to	O
the	O
XyGO	O
binding	O
site	O
in	O
SGBP	O
-	O
B	O
.	O
However	O
,	O
the	O
opposing	O
,	O
clamp	O
-	O
like	O
arrangement	O
of	O
these	O
residues	O
in	O
Bacova_04391	O
is	O
clearly	O
distinct	O
from	O
the	O
planar	O
surface	O
arrangement	O
of	O
the	O
residues	O
that	O
interact	O
with	O
XyG	O
in	O
SGBP	O
-	O
B	O
(	O
described	O
below	O
).	O
	O
Inspection	O
of	O
the	O
tertiary	O
structure	O
indicates	O
that	O
domains	O
C	O
and	O
D	O
are	O
effectively	O
inseparable	O
,	O
with	O
a	O
contact	O
interface	O
of	O
396	O
Å2	O
.	O
	O
Despite	O
the	O
lack	O
of	O
sequence	O
and	O
structural	O
conservation	O
,	O
a	O
similarly	O
positioned	O
proline	O
joins	O
the	O
Ig	O
-	O
like	O
domains	O
of	O
the	O
xylan	O
-	O
binding	O
Bacova_04391	O
and	O
the	O
starch	O
-	O
binding	O
proteins	O
SusE	O
and	O
SusF	O
.	O
We	O
speculate	O
that	O
this	O
is	O
a	O
biologically	O
important	O
adaptation	O
that	O
serves	O
to	O
project	O
the	O
glycan	O
binding	O
site	O
of	O
these	O
proteins	O
far	O
from	O
the	O
membrane	O
surface	O
.	O
	O
In	O
these	O
growth	O
experiments	O
,	O
overnight	O
cultures	O
of	O
strains	O
grown	O
on	O
minimal	O
medium	O
plus	O
glucose	O
were	O
back	O
-	O
diluted	O
1	O
:	O
100	O
-	O
fold	O
into	O
minimal	O
medium	O
containing	O
5	O
mg	O
/	O
ml	O
of	O
the	O
reported	O
carbohydrate	O
.	O
	O
Complementation	O
of	O
the	O
ΔSGBP	B-mutant
-	I-mutant
A	I-mutant
strain	O
(	O
ΔSGBP	B-mutant
-	I-mutant
A	I-mutant
::	O
SGBP	O
-	O
A	O
)	O
restores	O
growth	O
to	O
wild	O
-	O
type	O
rates	O
on	O
xyloglucan	O
and	O
XyGO1	O
,	O
yet	O
the	O
calculated	O
rate	O
of	O
the	O
complemented	O
strain	O
is	O
~	O
72	O
%	O
that	O
of	O
the	O
WT	O
Δtdk	B-mutant
strain	O
on	O
XyGO2	O
;	O
similar	O
results	O
were	O
obtained	O
for	O
the	O
SGBP	O
-	O
B	O
complemented	O
strain	O
despite	O
the	O
fact	O
that	O
the	O
growth	O
curves	O
do	O
not	O
appear	O
much	O
different	O
(	O
see	O
Fig	O
.	O
S8C	O
and	O
F	O
).	O
	O
Growth	O
was	O
measured	O
over	O
time	O
in	O
minimal	O
medium	O
containing	O
(	O
A	O
)	O
XyG	O
,	O
(	O
B	O
)	O
XyGO2	O
,	O
(	O
C	O
)	O
XyGO1	O
,	O
(	O
D	O
)	O
glucose	O
,	O
and	O
(	O
E	O
)	O
xylose	O
.	O
	O
In	O
panel	O
F	O
,	O
the	O
growth	O
rate	O
of	O
each	O
strain	O
on	O
the	O
five	O
carbon	O
sources	O
is	O
displayed	O
,	O
and	O
in	O
panel	O
G	O
,	O
the	O
normalized	O
lag	O
time	O
of	O
each	O
culture	O
,	O
relative	O
to	O
its	O
growth	O
on	O
glucose	O
,	O
is	O
displayed	O
.	O
	O
Intriguingly	O
,	O
the	O
ΔSGBP	B-mutant
-	I-mutant
B	I-mutant
strain	O
(	O
ΔBacova_02650	B-mutant
)	O
(	O
cf	O
.	O
	O
Fig	O
.	O
1B	O
)	O
exhibited	O
a	O
minor	O
growth	O
defect	O
on	O
both	O
XyG	O
and	O
XyGO2	O
,	O
with	O
rates	O
84	O
.	O
6	O
%	O
and	O
93	O
.	O
9	O
%	O
that	O
of	O
the	O
WT	O
Δtdk	B-mutant
strain	O
.	O
	O
However	O
,	O
growth	O
of	O
the	O
ΔSGBP	B-mutant
-	I-mutant
B	I-mutant
strain	O
on	O
XyGO1	O
was	O
54	O
.	O
2	O
%	O
the	O
rate	O
of	O
the	O
parental	O
strain	O
,	O
despite	O
the	O
fact	O
that	O
SGBP	O
-	O
B	O
binds	O
this	O
substrate	O
ca	O
.	O
	O
Taken	O
together	O
,	O
the	O
data	O
indicate	O
that	O
SGBP	O
-	O
A	O
and	O
SGBP	O
-	O
B	O
functionally	O
complement	O
each	O
other	O
in	O
the	O
capture	O
of	O
XyG	O
polysaccharide	O
,	O
while	O
SGBP	O
-	O
B	O
may	O
allow	O
B	O
.	O
ovatus	O
to	O
scavenge	O
smaller	O
XyGOs	O
liberated	O
by	O
other	O
gut	O
commensals	O
.	O
	O
It	O
may	O
then	O
be	O
that	O
only	O
after	O
a	O
sufficient	O
amount	O
of	O
glycan	O
is	O
processed	O
and	O
imported	O
by	O
the	O
cell	O
is	O
XyGUL	O
upregulated	O
and	O
exponential	O
growth	O
on	O
the	O
glycan	O
can	O
begin	O
.	O
	O
Likewise	O
,	O
such	O
cognate	O
interactions	O
between	O
homologous	O
protein	O
pairs	O
such	O
as	O
SGBP	O
-	O
A	O
and	O
its	O
TBDT	O
may	O
underlie	O
our	O
observation	O
that	O
a	O
ΔSGBP	B-mutant
-	I-mutant
A	I-mutant
mutant	O
cannot	O
grow	O
on	O
xyloglucan	O
.	O
	O
Thus	O
,	O
understanding	O
glycan	O
capture	O
at	O
the	O
cell	O
surface	O
is	O
fundamental	O
to	O
explaining	O
,	O
and	O
eventually	O
predicting	O
,	O
how	O
the	O
carbohydrate	O
content	O
of	O
the	O
diet	O
shapes	O
the	O
gut	O
community	O
structure	O
as	O
well	O
as	O
its	O
causative	O
health	O
effects	O
.	O
	O
PUL	O
-	O
encoded	O
TBDTs	O
in	O
Bacteroidetes	O
are	O
larger	O
than	O
the	O
well	O
-	O
characterized	O
iron	O
-	O
targeting	O
TBDTs	O
from	O
many	O
Proteobacteria	O
and	O
are	O
further	O
distinguished	O
as	O
the	O
only	O
known	O
glycan	O
-	O
importing	O
TBDTs	O
coexpressed	O
with	O
an	O
SGBP	O
.	O
	O
Our	O
observation	O
here	O
that	O
the	O
physical	O
presence	O
of	O
the	O
SusD	O
homolog	O
SGBP	O
-	O
A	O
,	O
independent	O
of	O
XyG	O
-	O
binding	O
ability	O
,	O
is	O
both	O
necessary	O
and	O
sufficient	O
for	O
XyG	O
utilization	O
further	O
supports	O
a	O
model	O
of	O
glycan	O
import	O
whereby	O
the	O
SusC	O
-	O
like	O
TBDTs	O
and	O
the	O
SusD	O
-	O
like	O
SGBPs	O
must	O
be	O
intimately	O
associated	O
to	O
support	O
glycan	O
uptake	O
(	O
Fig	O
.	O
1C	O
).	O
	O
A	O
molecular	O
understanding	O
of	O
glycan	O
uptake	O
by	O
human	O
gut	O
bacteria	O
is	O
therefore	O
central	O
to	O
the	O
development	O
of	O
strategies	O
to	O
improve	O
human	O
health	O
through	O
manipulation	O
of	O
the	O
microbiota	O
.	O
	O
A	O
high	O
affinity	O
IL	O
-	O
17A	O
peptide	O
antagonist	O
(	O
HAP	O
)	O
of	O
15	O
residues	O
was	O
identified	O
through	O
phage	O
-	O
display	O
screening	O
followed	O
by	O
saturation	O
mutagenesis	O
optimization	O
and	O
amino	O
acid	O
substitutions	O
.	O
	O
The	O
family	O
of	O
IL	O
-	O
17	O
cytokines	O
and	O
receptors	O
consists	O
of	O
six	O
polypeptides	O
,	O
IL	O
-	O
17A	O
-	O
F	O
,	O
and	O
five	O
receptors	O
,	O
IL	O
-	O
17RA	O
-	O
E	O
.	O
IL	O
-	O
17A	O
is	O
secreted	O
from	O
activated	O
Th17	O
cells	O
,	O
and	O
several	O
innate	O
immune	O
T	O
cell	O
types	O
including	O
macrophages	O
,	O
neutrophils	O
,	O
natural	O
killer	O
cells	O
,	O
and	O
dendritic	O
cells	O
.	O
	O
There	O
has	O
been	O
active	O
research	O
in	O
identifying	O
orally	O
available	O
chemical	O
entities	O
that	O
would	O
functionally	O
antagonize	O
IL	O
-	O
17A	O
-	O
mediated	O
signaling	O
.	O
	O
Since	O
IL	O
-	O
17RA	O
is	O
a	O
shared	O
receptor	O
for	O
at	O
least	O
IL	O
-	O
17A	O
,	O
IL	O
-	O
17F	O
,	O
IL	O
-	O
17A	O
/	O
IL	O
-	O
17F	O
and	O
IL	O
-	O
17E	O
,	O
we	O
chose	O
to	O
seek	O
IL	O
-	O
17A	O
-	O
specific	O
inhibitors	O
that	O
may	O
have	O
more	O
defined	O
pharmacological	O
responses	O
than	O
IL	O
-	O
17RA	O
inhibitors	O
.	O
	O
Positive	O
phage	O
pools	O
were	O
then	O
sub	O
-	O
cloned	O
into	O
a	O
maltose	O
-	O
binding	O
protein	O
(	O
MBP	O
)	O
fusion	O
system	O
.	O
	O
Sequences	O
identified	O
from	O
phage	O
clones	O
were	O
chemically	O
synthesized	O
(	O
Supplementary	O
Table	O
1	O
)	O
and	O
tested	O
for	O
inhibition	O
of	O
IL	O
-	O
17A	O
binding	O
to	O
IL	O
-	O
17RA	O
(	O
Table	O
1	O
).	O
	O
In	O
particular	O
,	O
at	O
position	O
5	O
(	O
13	O
),	O
substitution	O
of	O
methionine	O
with	O
alanine	O
resulted	O
in	O
a	O
seven	O
fold	O
improvement	O
in	O
potency	O
(	O
80	O
nM	O
versus	O
11	O
nM	O
respectively	O
).	O
	O
Since	O
the	O
replacement	O
of	O
methionine	O
at	O
position	O
5	O
with	O
alanine	O
was	O
beneficial	O
,	O
the	O
additional	O
hydrophobic	O
amino	O
acids	O
isoleucine	O
(	O
24	O
),	O
leucine	O
(	O
25	O
)	O
and	O
valine	O
(	O
26	O
)	O
were	O
evaluated	O
and	O
an	O
additional	O
two	O
-	O
three	O
fold	O
improvement	O
in	O
binding	O
was	O
observed	O
for	O
the	O
valine	O
and	O
isoleucine	O
replacements	O
in	O
comparison	O
with	O
alanine	O
.	O
	O
Dimerization	O
of	O
HAP	O
can	O
further	O
increase	O
its	O
potency	O
	O
Orthogonal	O
assays	O
to	O
confirm	O
HAP	O
antagonism	O
	O
The	O
relatively	O
high	O
IC50	O
values	O
in	O
this	O
assay	O
(	O
Table	O
3	O
)	O
are	O
probably	O
due	O
to	O
the	O
high	O
IL	O
-	O
17A	O
concentration	O
(	O
100	O
ng	O
/	O
ml	O
)	O
needed	O
for	O
detection	O
of	O
IL	O
-	O
6	O
.	O
	O
Crystallization	O
and	O
structure	O
determination	O
	O
Crystals	O
of	O
the	O
Fab	O
/	O
truncated	O
IL	O
-	O
17A	O
/	O
HAP	O
complex	O
diffracted	O
to	O
2	O
.	O
2	O
Å	O
,	O
and	O
the	O
Fab	O
/	O
full	O
length	O
IL	O
-	O
17A	O
/	O
HAP	O
complex	O
diffracted	O
to	O
3	O
.	O
0	O
Å	O
(	O
Supplementary	O
Table	O
S3	O
).	O
	O
Two	O
copies	O
of	O
HAP	O
bind	O
to	O
the	O
N	O
-	O
terminal	O
of	O
the	O
cytokine	O
dimer	O
,	O
also	O
symmetrically	O
,	O
and	O
each	O
HAP	O
molecule	O
also	O
interacts	O
with	O
both	O
IL	O
-	O
17A	O
monomers	O
(	O
Fig	O
.	O
2	O
).	O
	O
Inhibition	O
mechanism	O
of	O
IL	O
-	O
17A	O
signaling	O
by	O
HAP	O
	O
Structure	O
basis	O
for	O
the	O
observed	O
SAR	O
of	O
peptides	O
	O
The	O
C	O
-	O
terminal	O
Asn14	O
and	O
Lys15	O
of	O
HAP	O
are	O
not	O
directly	O
involved	O
in	O
interactions	O
with	O
IL	O
-	O
17A	O
,	O
and	O
this	O
is	O
reflected	O
in	O
the	O
gradual	O
reduction	O
in	O
activity	O
caused	O
by	O
C	O
-	O
terminal	O
truncations	O
(	O
35	O
and	O
36	O
,	O
Table	O
2	O
).	O
	O
For	O
example	O
,	O
inspection	O
of	O
the	O
published	O
IL	O
-	O
17F	O
crystal	O
structure	O
(	O
PDB	O
code	O
1JPY	O
)	O
revealed	O
a	O
pocket	O
of	O
IL	O
-	O
17F	O
similar	O
to	O
that	O
of	O
IL	O
-	O
17A	O
for	O
W12	O
of	O
HAP	O
binding	O
,	O
but	O
it	O
is	O
occupied	O
by	O
a	O
Phe	O
-	O
Phe	O
motif	O
at	O
the	O
N	O
-	O
terminal	O
peptide	O
of	O
IL	O
-	O
17F	O
.	O
	O
We	O
have	O
also	O
determined	O
the	O
complex	O
structure	O
of	O
IL	O
-	O
17A	O
/	O
HAP	O
,	O
which	O
provides	O
the	O
structural	O
basis	O
for	O
HAP	O
’	O
s	O
antagonism	O
to	O
IL	O
-	O
17A	O
signaling	O
.	O
	O
Since	O
apo	O
IL	O
-	O
17A	O
is	O
a	O
homodimer	O
with	O
2	O
fold	O
symmetry	O
,	O
IL	O
-	O
17RA	O
potentially	O
can	O
bind	O
to	O
either	O
face	O
of	O
the	O
IL	O
-	O
17A	O
dimer	O
.	O
	O
The	O
interaction	O
of	O
IL	O
-	O
17A	O
with	O
IL	O
-	O
17RA	O
has	O
an	O
extensive	O
interface	O
,	O
covering	O
~	O
2	O
,	O
200	O
Å2	O
surface	O
area	O
of	O
IL	O
-	O
17A	O
.	O
	O
One	O
way	O
of	O
further	O
improving	O
HAP	O
’	O
s	O
potency	O
is	O
by	O
dimerization	O
.	O
	O
KD	O
determined	O
by	O
the	O
standard	O
equation	O
,	O
KD	O
=	O
kd	O
/	O
ka	O
.	O
(	O
B	O
)	O
HAP	O
inhibits	O
SPR	O
signaling	O
of	O
IL	O
-	O
17A	O
binding	O
to	O
immobilized	O
IL	O
-	O
17RA	O
.	O
	O
Overall	O
structure	O
of	O
the	O
Fab	O
/	O
IL	O
-	O
17A	O
/	O
HAP	O
complex	O
in	O
ribbon	O
presentation	O
.	O
	O
(	O
C	O
)	O
As	O
a	O
comparison	O
,	O
the	O
IL	O
-	O
17A	O
/	O
IL	O
-	O
17RA	O
complex	O
was	O
shown	O
with	O
IL	O
-	O
17A	O
in	O
the	O
same	O
orientation	O
.	O
	O
ELISA	O
competition	O
activity	O
of	O
peptide	O
analogues	O
of	O
1	O
.	O
	O
The	O
amount	O
of	O
NadA	O
on	O
the	O
bacterial	O
surface	O
is	O
of	O
direct	O
relevance	O
in	O
the	O
constant	O
battle	O
of	O
host	O
-	O
pathogen	O
interactions	O
:	O
it	O
influences	O
the	O
ability	O
of	O
the	O
pathogen	O
to	O
engage	O
human	O
cell	O
surface	O
-	O
exposed	O
receptors	O
and	O
,	O
conversely	O
,	O
the	O
bacterial	O
susceptibility	O
to	O
the	O
antibody	O
-	O
mediated	O
immune	O
response	O
.	O
	O
NadR	O
also	O
mediates	O
ligand	O
-	O
dependent	O
regulation	O
of	O
many	O
other	O
meningococcal	O
genes	O
,	O
for	O
example	O
the	O
highly	O
-	O
conserved	O
multiple	O
adhesin	O
family	O
(	O
maf	O
)	O
genes	O
,	O
which	O
encode	O
proteins	O
emerging	O
with	O
important	O
roles	O
in	O
host	O
-	O
pathogen	O
interactions	O
,	O
immune	O
evasion	O
and	O
niche	O
adaptation	O
.	O
	O
The	O
abundance	O
of	O
surface	O
-	O
exposed	O
NadA	O
is	O
regulated	O
by	O
the	O
ligand	O
-	O
responsive	O
transcriptional	O
repressor	O
NadR	O
.	O
Here	O
,	O
we	O
present	O
functional	O
,	O
biochemical	O
and	O
high	O
-	O
resolution	O
structural	O
data	O
on	O
NadR	O
.	O
Our	O
studies	O
provide	O
detailed	O
insights	O
into	O
how	O
small	O
molecule	O
ligands	O
,	O
such	O
as	O
hydroxyphenylacetate	O
derivatives	O
,	O
found	O
in	O
relevant	O
host	O
niches	O
,	O
modulate	O
the	O
structure	O
and	O
activity	O
of	O
NadR	O
,	O
by	O
‘	O
conformational	O
selection	O
’	O
of	O
inactive	O
forms	O
.	O
	O
The	O
DNA	O
-	O
binding	O
activity	O
of	O
NadR	O
is	O
attenuated	O
in	O
vitro	O
upon	O
addition	O
of	O
various	O
hydroxyphenylacetate	O
(	O
HPA	O
)	O
derivatives	O
,	O
including	O
4	O
-	O
HPA	O
.	O
	O
Moreover	O
,	O
these	O
findings	O
are	O
important	O
because	O
the	O
activity	O
of	O
NadR	O
impacts	O
the	O
potential	O
coverage	O
provided	O
by	O
anti	O
-	O
NadA	O
antibodies	O
elicited	O
by	O
the	O
Bexsero	O
vaccine	O
and	O
influences	O
host	O
-	O
bacteria	O
interactions	O
that	O
contribute	O
to	O
meningococcal	O
pathogenesis	O
.	O
	O
In	O
analytical	O
size	O
-	O
exclusion	O
high	O
-	O
performance	O
liquid	O
chromatography	O
(	O
SE	O
-	O
HPLC	O
)	O
experiments	O
coupled	O
with	O
multi	O
-	O
angle	O
laser	O
light	O
scattering	O
(	O
MALLS	O
),	O
NadR	O
presented	O
a	O
single	O
species	O
with	O
an	O
absolute	O
molecular	O
mass	O
of	O
35	O
kDa	O
(	O
S1	O
Fig	O
).	O
	O
(	O
A	O
)	O
Molecular	O
structures	O
of	O
3	O
-	O
HPA	O
(	O
MW	O
152	O
.	O
2	O
),	O
4	O
-	O
HPA	O
(	O
MW	O
152	O
.	O
2	O
),	O
3Cl	O
,	O
4	O
-	O
HPA	O
(	O
MW	O
186	O
.	O
6	O
)	O
and	O
salicylic	O
acid	O
(	O
MW	O
160	O
.	O
1	O
).	O
(	O
B	O
)	O
DSC	O
profiles	O
,	O
colored	O
as	O
follows	O
:	O
apo	O
-	O
NadR	O
(	O
violet	O
),	O
NadR	O
+	O
salicylate	O
(	O
red	O
),	O
NadR	O
+	O
3	O
-	O
HPA	O
(	O
green	O
),	O
NadR	O
+	O
4	O
-	O
HPA	O
(	O
blue	O
),	O
NadR	O
+	O
3Cl	O
,	O
4	O
-	O
HPA	O
(	O
pink	O
).	O
	O
All	O
DSC	O
profiles	O
are	O
representative	O
of	O
triplicate	O
experiments	O
.	O
	O
However	O
,	O
steady	O
-	O
state	O
SPR	O
analyses	O
of	O
the	O
NadR	O
-	O
HPA	O
interactions	O
allowed	O
determination	O
of	O
the	O
equilibrium	O
dissociation	O
constants	O
(	O
KD	O
)	O
(	O
Table	O
1	O
and	O
S2	O
Fig	O
).	O
	O
The	O
interactions	O
of	O
4	O
-	O
HPA	O
and	O
3Cl	O
,	O
4	O
-	O
HPA	O
with	O
NadR	O
exhibited	O
KD	O
values	O
of	O
1	O
.	O
5	O
mM	O
and	O
1	O
.	O
1	O
mM	O
,	O
respectively	O
.	O
	O
To	O
fully	O
characterize	O
the	O
NadR	O
/	O
HPA	O
interactions	O
,	O
we	O
sought	O
to	O
determine	O
crystal	O
structures	O
of	O
NadR	O
in	O
ligand	O
-	O
bound	O
(	O
holo	O
)	O
and	O
ligand	O
-	O
free	O
(	O
apo	O
)	O
forms	O
.	O
	O
The	O
map	O
is	O
contoured	O
at	O
1σ	O
and	O
the	O
figure	O
was	O
prepared	O
with	O
a	O
density	O
mesh	O
carve	O
factor	O
of	O
1	O
.	O
7	O
,	O
using	O
Pymol	O
(	O
www	O
.	O
pymol	O
.	O
org	O
).	O
	O
Only	O
the	O
mutation	O
L130K	B-mutant
has	O
a	O
noteworthy	O
effect	O
on	O
the	O
oligomeric	O
state	O
,	O
inducing	O
a	O
second	O
peak	O
with	O
a	O
longer	O
retention	O
time	O
and	O
a	O
second	O
peak	O
maximum	O
at	O
18	O
.	O
6	O
min	O
.	O
	O
To	O
a	O
much	O
lesser	O
extent	O
,	O
the	O
L133K	B-mutant
mutation	O
also	O
appears	O
to	O
induce	O
a	O
‘	O
shoulder	O
’	O
to	O
the	O
main	O
peak	O
,	O
suggesting	O
very	O
weak	O
ability	O
to	O
disrupt	O
the	O
dimer	O
.	O
(	O
D	O
)	O
SE	O
-	O
HPLC	O
/	O
MALLS	O
analyses	O
of	O
the	O
L130K	B-mutant
mutant	O
,	O
shows	O
20	O
%	O
dimer	O
and	O
80	O
%	O
monomer	O
.	O
	O
The	O
ligand	O
showed	O
a	O
different	O
position	O
and	O
orientation	O
compared	O
to	O
salicylate	O
complexed	O
with	O
MTH313	O
and	O
ST1710	O
(	O
see	O
Discussion	O
).	O
	O
At	O
the	O
other	O
‘	O
end	O
’	O
of	O
the	O
ligand	O
,	O
the	O
4	O
-	O
hydroxyl	O
group	O
was	O
proximal	O
to	O
AspB36	O
,	O
with	O
which	O
it	O
may	O
establish	O
an	O
H	O
-	O
bond	O
(	O
see	O
bond	O
distances	O
in	O
Table	O
3	O
).	O
	O
The	O
water	O
molecule	O
observed	O
in	O
the	O
pocket	O
was	O
bound	O
by	O
the	O
carboxylate	O
group	O
and	O
the	O
side	O
chains	O
of	O
SerA9	O
and	O
AsnA11	O
.	O
	O
In	O
addition	O
to	O
the	O
H	O
-	O
bonds	O
involving	O
the	O
carboxylate	O
and	O
hydroxyl	O
groups	O
of	O
4	O
-	O
HPA	O
,	O
binding	O
of	O
the	O
phenyl	O
moiety	O
appeared	O
to	O
be	O
stabilized	O
by	O
several	O
van	O
der	O
Waals	O
’	O
contacts	O
,	O
particularly	O
those	O
involving	O
the	O
hydrophobic	O
side	O
chain	O
atoms	O
of	O
LeuB21	O
,	O
MetB22	O
,	O
PheB25	O
,	O
LeuB29	O
and	O
ValB111	O
(	O
Fig	O
4A	O
).	O
	O
The	O
presence	O
of	O
a	O
single	O
hydroxyl	O
group	O
at	O
position	O
2	O
,	O
as	O
in	O
2	O
-	O
HPA	O
,	O
rather	O
than	O
at	O
position	O
4	O
,	O
would	O
eliminate	O
the	O
possibility	O
of	O
favorable	O
interactions	O
with	O
AspB36	O
,	O
resulting	O
in	O
the	O
lack	O
of	O
NadR	O
regulation	O
by	O
2	O
-	O
HPA	O
described	O
previously	O
.	O
	O
Firstly	O
,	O
NadR	O
is	O
expected	O
to	O
be	O
covalently	O
immobilized	O
on	O
the	O
sensor	O
chip	O
as	O
a	O
dimer	O
in	O
random	O
orientations	O
,	O
since	O
it	O
is	O
a	O
stable	O
dimer	O
in	O
solution	O
and	O
has	O
sixteen	O
lysines	O
well	O
-	O
distributed	O
around	O
its	O
surface	O
,	O
all	O
able	O
to	O
act	O
as	O
potential	O
sites	O
for	O
amine	O
coupling	O
to	O
the	O
chip	O
,	O
and	O
none	O
of	O
which	O
are	O
close	O
to	O
the	O
ligand	O
-	O
binding	O
pocket	O
.	O
	O
Secondly	O
,	O
the	O
HPA	O
analytes	O
are	O
all	O
very	O
small	O
(	O
MW	O
150	O
–	O
170	O
,	O
Fig	O
1A	O
)	O
and	O
therefore	O
are	O
expected	O
to	O
be	O
able	O
to	O
diffuse	O
readily	O
into	O
all	O
potential	O
binding	O
sites	O
,	O
irrespective	O
of	O
the	O
random	O
orientations	O
of	O
the	O
immobilized	O
NadR	O
dimers	O
on	O
the	O
chip	O
.	O
	O
The	O
crystallographic	O
data	O
,	O
supported	O
by	O
the	O
SPR	O
studies	O
of	O
binding	O
stoichiometry	O
,	O
revealed	O
the	O
lack	O
of	O
a	O
second	O
4	O
-	O
HPA	O
molecule	O
in	O
the	O
homodimer	O
,	O
suggesting	O
negative	O
co	O
-	O
operativity	O
,	O
a	O
phenomenon	O
previously	O
described	O
for	O
the	O
MTH313	O
/	O
salicylate	O
interaction	O
and	O
for	O
other	O
MarR	O
family	O
proteins	O
.	O
	O
To	O
explore	O
the	O
molecular	O
basis	O
of	O
asymmetry	O
in	O
holo	O
-	O
NadR	O
,	O
we	O
superposed	O
its	O
ligand	O
-	O
free	O
monomer	O
(	O
chain	O
A	O
)	O
onto	O
the	O
ligand	O
-	O
occupied	O
monomer	O
(	O
chain	O
B	O
).	O
	O
However	O
,	O
since	O
residues	O
of	O
helix	O
α6	O
were	O
not	O
directly	O
involved	O
in	O
ligand	O
binding	O
,	O
an	O
explanation	O
for	O
the	O
lack	O
of	O
4	O
-	O
HPA	O
in	O
monomer	O
A	O
did	O
not	O
emerge	O
by	O
analyzing	O
only	O
these	O
backbone	O
atom	O
positions	O
,	O
suggesting	O
that	O
a	O
more	O
complex	O
series	O
of	O
allosteric	O
events	O
may	O
occur	O
.	O
	O
Specifically	O
,	O
upon	O
analysis	O
with	O
the	O
CASTp	O
software	O
,	O
the	O
pocket	O
in	O
chain	O
B	O
containing	O
the	O
4	O
-	O
HPA	O
exhibited	O
a	O
total	O
volume	O
of	O
approximately	O
370	O
Å3	O
,	O
while	O
the	O
pocket	O
in	O
chain	O
A	O
was	O
occupied	O
by	O
these	O
three	O
side	O
chains	O
that	O
adopted	O
‘	O
inward	O
’	O
positions	O
and	O
thereby	O
divided	O
the	O
space	O
into	O
a	O
few	O
much	O
smaller	O
pockets	O
,	O
each	O
with	O
volume	O
<	O
50	O
Å3	O
,	O
evidently	O
rendering	O
chain	O
A	O
unfavorable	O
for	O
ligand	O
binding	O
.	O
	O
Although	O
more	O
comprehensive	O
NMR	O
experiments	O
and	O
full	O
chemical	O
shift	O
assignment	O
of	O
the	O
spectra	O
would	O
be	O
required	O
to	O
precisely	O
define	O
this	O
multi	O
-	O
state	O
behavior	O
,	O
the	O
NMR	O
data	O
clearly	O
demonstrate	O
that	O
NadR	O
exhibits	O
conformational	O
flexibility	O
which	O
is	O
modulated	O
by	O
4	O
-	O
HPA	O
in	O
solution	O
.	O
	O
(	O
A	O
)	O
The	O
holo	O
-	O
homodimer	O
structure	O
is	O
shown	O
as	O
green	O
and	O
blue	O
cartoons	O
,	O
for	O
chain	O
A	O
and	O
B	O
,	O
respectively	O
,	O
while	O
the	O
two	O
homodimers	O
of	O
apo	O
-	O
NadR	O
are	O
both	O
cyan	O
and	O
pale	O
blue	O
for	O
chains	O
A	O
/	O
C	O
and	O
B	O
/	O
D	O
,	O
respectively	O
.	O
	O
The	O
three	O
homodimers	O
(	O
chains	O
AB	O
holo	O
,	O
AB	O
apo	O
,	O
and	O
CD	O
apo	O
)	O
were	O
overlaid	O
by	O
structural	O
alignment	O
exclusively	O
of	O
all	O
heavy	O
atoms	O
in	O
residues	O
R64	O
-	O
A77	O
(	O
shown	O
in	O
red	O
,	O
with	O
side	O
chain	O
sticks	O
)	O
of	O
chains	O
A	O
holo	O
,	O
A	O
apo	O
,	O
and	O
C	O
apo	O
,	O
belonging	O
to	O
helix	O
α4	O
(	O
left	O
).	O
	O
Thus	O
,	O
the	O
apo	O
-	O
homodimer	O
AB	O
presented	O
the	O
DNA	O
-	O
binding	O
helices	O
in	O
a	O
conformation	O
similar	O
to	O
that	O
observed	O
in	O
the	O
protein	O
:	O
DNA	O
complex	O
of	O
OhrR	O
:	O
ohrA	O
from	O
Bacillus	O
subtilis	O
(	O
Fig	O
8C	O
).	O
	O
This	O
mutagenesis	O
data	O
revealed	O
that	O
NadR	O
residues	O
His7	O
,	O
Ser9	O
,	O
Asn11	O
and	O
Phe25	O
play	O
key	O
roles	O
in	O
the	O
ligand	O
-	O
mediated	O
regulation	O
of	O
NadR	O
;	O
they	O
are	O
each	O
involved	O
in	O
the	O
controlled	O
de	O
-	O
repression	O
of	O
the	O
nadA	O
promoter	O
and	O
synthesis	O
of	O
NadA	O
in	O
response	O
to	O
4	O
-	O
HPA	O
in	O
vivo	O
.	O
	O
Given	O
the	O
importance	O
of	O
NadR	O
-	O
mediated	O
regulation	O
of	O
NadA	O
levels	O
in	O
the	O
contexts	O
of	O
meningococcal	O
pathogenesis	O
,	O
we	O
sought	O
to	O
characterize	O
NadR	O
,	O
and	O
its	O
interaction	O
with	O
ligands	O
,	O
at	O
atomic	O
resolution	O
.	O
	O
(	O
B	O
)	O
A	O
structural	O
alignment	O
of	O
MTH313	O
chain	O
A	O
and	O
ST1710	O
(	O
pink	O
)	O
(	O
Cα	O
rmsd	O
2	O
.	O
3Å	O
),	O
shows	O
that	O
they	O
bind	O
salicylate	O
in	O
equivalent	O
sites	O
(	O
differing	O
by	O
only	O
~	O
3Å	O
)	O
and	O
with	O
the	O
same	O
orientation	O
.	O
	O
While	O
some	O
flexibility	O
of	O
helix	O
α4	O
was	O
also	O
observed	O
in	O
the	O
two	O
apo	O
-	O
structures	O
,	O
concomitant	O
changes	O
in	O
the	O
dimer	O
interfaces	O
were	O
not	O
observed	O
,	O
possibly	O
due	O
to	O
the	O
absence	O
of	O
ligand	O
.	O
	O
The	O
latter	O
may	O
influence	O
the	O
surface	O
abundance	O
or	O
secretion	O
of	O
maf	O
proteins	O
,	O
an	O
emerging	O
class	O
of	O
highly	O
conserved	O
meningococcal	O
putative	O
adhesins	O
and	O
toxins	O
with	O
many	O
important	O
roles	O
.	O
	O
Further	O
work	O
is	O
required	O
to	O
investigate	O
how	O
the	O
two	O
different	O
promoter	O
types	O
influence	O
the	O
ligand	O
-	O
responsiveness	O
of	O
NadR	O
during	O
bacterial	O
infection	O
and	O
may	O
provide	O
insights	O
into	O
the	O
regulatory	O
mechanisms	O
occurring	O
during	O
these	O
host	O
-	O
pathogen	O
interactions	O
.	O
	O
Structure	O
of	O
an	O
OhrR	O
-	O
ohrA	O
operator	O
complex	O
reveals	O
the	O
DNA	O
binding	O
mechanism	O
of	O
the	O
MarR	O
family	O
	O
Structural	O
determinant	O
for	O
inducing	O
RORgamma	O
specific	O
inverse	O
agonism	O
triggered	O
by	O
a	O
synthetic	O
benzoxazinone	O
ligand	O
	O
Our	O
goal	O
was	O
to	O
develop	O
a	O
RORγ	O
specific	O
inverse	O
agonist	O
that	O
would	O
help	O
down	O
regulate	O
pro	O
-	O
inflammatory	O
gene	O
transcription	O
by	O
disrupting	O
the	O
protein	O
protein	O
interaction	O
with	O
coactivator	O
proteins	O
as	O
a	O
therapeutic	O
agent	O
.	O
	O
Using	O
an	O
in	O
vivo	O
reporter	O
assay	O
,	O
we	O
show	O
that	O
the	O
inverse	O
agonist	O
BIO399	O
displayed	O
specificity	O
for	O
RORγ	O
over	O
ROR	O
sub	O
-	O
family	O
members	O
α	O
and	O
β	O
.	O
	O
The	O
synthetic	O
benzoxazinone	O
ligands	O
identified	O
in	O
our	O
FRET	O
assay	O
have	O
an	O
agonist	O
(	O
BIO592	O
)	O
or	O
inverse	O
agonist	O
(	O
BIO399	O
)	O
effect	O
by	O
stabilizing	O
or	O
destabilizing	O
the	O
agonist	O
conformation	O
of	O
RORγ	O
.	O
	O
Our	O
structural	O
investigation	O
of	O
the	O
BIO592	O
agonist	O
and	O
BIO399	O
inverse	O
agonist	O
structures	O
identified	O
residue	O
Met358	O
on	O
RORγ	O
as	O
the	O
trigger	O
for	O
RORγ	O
specific	O
inverse	O
agonism	O
.	O
	O
Retinoid	O
-	O
related	O
orphan	O
receptor	O
gamma	O
(	O
RORγ	O
)	O
is	O
a	O
transcription	O
factor	O
belonging	O
to	O
a	O
sub	O
-	O
family	O
of	O
nuclear	O
receptors	O
that	O
includes	O
two	O
closely	O
related	O
members	O
RORα	O
and	O
RORβ	O
.	O
	O
Here	O
we	O
present	O
the	O
identification	O
of	O
two	O
synthetic	O
benzoxazinone	O
RORγ	O
ligands	O
,	O
a	O
weak	O
agonist	O
BIO592	O
(	O
Fig	O
.	O
1a	O
)	O
and	O
an	O
inverse	O
agonist	O
BIO399	O
(	O
Fig	O
.	O
1b	O
)	O
which	O
were	O
identified	O
using	O
a	O
Fluorescence	O
Resonance	O
Energy	O
transfer	O
(	O
FRET	O
)	O
based	O
assay	O
that	O
monitored	O
coactivator	O
peptide	O
recruitment	O
.	O
	O
Using	O
partial	O
proteolysis	O
in	O
combination	O
with	O
mass	O
spectrometry	O
analysis	O
we	O
demonstrate	O
that	O
the	O
AF2	O
helix	O
of	O
RORγ	O
destabilizes	O
upon	O
BIO399	O
(	O
inverse	O
agonist	O
)	O
binding	O
.	O
	O
Using	O
a	O
FRET	O
based	O
assay	O
we	O
discovered	O
agonist	O
BIO592	O
(	O
Fig	O
.	O
1a	O
)	O
which	O
increased	O
the	O
coactivator	O
peptide	O
TRAP220	O
recruitment	O
to	O
RORγ	O
(	O
EC50	O
0f	O
58nM	O
and	O
Emax	O
of	O
130	O
%)	O
and	O
a	O
potent	O
inverse	O
agonist	O
BIO399	O
(	O
Fig	O
.	O
1b	O
)	O
which	O
inhibited	O
coactivator	O
recruitment	O
(	O
IC50	O
:	O
4	O
.	O
7nM	O
).	O
	O
c	O
EBI96	O
coactivator	O
peptide	O
bound	O
in	O
the	O
coactivator	O
pocket	O
of	O
RORγ	O
	O
Specific	O
proteolytic	O
positions	O
on	O
RORγ518	O
when	O
treated	O
with	O
Actinase	O
E	O
alone	O
(	O
Green	O
)	O
or	O
in	O
the	O
presence	O
of	O
BIO399	O
(	O
Red	O
)	O
and	O
shared	O
proteolytic	O
sites	O
(	O
Yellow	O
)	O
	O
Several	O
rounds	O
of	O
cocrystallization	O
attempts	O
with	O
RORγ518	O
or	O
other	O
RORγ	O
AF2	O
helix	O
containing	O
constructs	O
complexed	O
with	O
BIO399	O
had	O
not	O
produced	O
crystals	O
.	O
	O
We	O
reasoned	O
that	O
if	O
we	O
could	O
remove	O
the	O
unfolded	O
AF2	O
helix	O
using	O
proteolysis	O
we	O
could	O
produce	O
a	O
binary	O
complex	O
more	O
amenable	O
to	O
crystallization	O
.	O
	O
The	O
aeRORγ493	O
/	O
4	O
BIO399	O
structure	O
diverged	O
at	O
the	O
c	O
-	O
terminal	O
end	O
of	O
Helix	O
11	O
from	O
the	O
RORγ518	O
BIO592	O
EBI96	O
structure	O
,	O
where	O
helix	O
11	O
unwinds	O
into	O
a	O
random	O
coil	O
after	O
residue	O
L475	O
.	O
	O
BIO399	O
binds	O
to	O
the	O
ligand	O
binding	O
site	O
of	O
RORγ	O
adopting	O
a	O
collapsed	O
conformation	O
as	O
seen	O
with	O
BIO592	O
where	O
the	O
two	O
compounds	O
superimpose	O
with	O
an	O
RMSD	O
of	O
0	O
.	O
72	O
Å	O
(	O
Fig	O
.	O
5b	O
).	O
	O
BIO399	O
and	O
Inverse	O
agonist	O
T0901317	O
bind	O
in	O
a	O
collapsed	O
conformation	O
distinct	O
from	O
other	O
RORγ	O
Inverse	O
Agonists	O
Cocrystal	O
structures	O
	O
However	O
,	O
the	O
inverse	O
agonism	O
trigger	O
of	O
BIO399	O
,	O
residue	O
Met358	O
,	O
is	O
a	O
leucine	O
in	O
both	O
RORα	O
and	O
β	O
.	O
	O
The	O
Structural	O
Basis	O
of	O
Coenzyme	O
A	O
Recycling	O
in	O
a	O
Bacterial	O
Organelle	O
	O
The	O
majority	O
of	O
catabolic	O
BMCs	O
(	O
metabolosomes	O
)	O
compartmentalize	O
a	O
common	O
core	O
of	O
enzymes	O
to	O
metabolize	O
compounds	O
via	O
a	O
toxic	O
and	O
/	O
or	O
volatile	O
aldehyde	O
intermediate	O
.	O
	O
Accordingly	O
,	O
PduL	O
and	O
Pta	O
exemplify	O
functional	O
,	O
but	O
not	O
structural	O
,	O
convergent	O
evolution	O
.	O
	O
This	O
enzyme	O
,	O
PduL	O
,	O
is	O
exclusively	O
associated	O
with	O
organelles	O
called	O
bacterial	O
microcompartments	O
,	O
which	O
are	O
used	O
to	O
catabolize	O
various	O
compounds	O
.	O
	O
The	O
aldehyde	O
is	O
subsequently	O
converted	O
into	O
an	O
acyl	O
-	O
CoA	O
by	O
aldehyde	O
dehydrogenase	O
,	O
which	O
uses	O
NAD	O
+	O
and	O
CoA	O
as	O
cofactors	O
.	O
	O
NAD	O
+	O
is	O
recycled	O
via	O
alcohol	O
dehydrogenase	O
,	O
and	O
CoA	O
is	O
recycled	O
via	O
phosphotransacetylase	O
(	O
PTAC	O
)	O
(	O
Fig	O
1	O
).	O
	O
They	O
can	O
also	O
work	O
in	O
the	O
reverse	O
direction	O
to	O
activate	O
acetate	O
to	O
the	O
CoA	O
-	O
thioester	O
.	O
	O
The	O
canonical	O
PTAC	O
,	O
Pta	O
,	O
is	O
an	O
ancient	O
enzyme	O
found	O
in	O
some	O
eukaryotes	O
and	O
archaea	O
,	O
and	O
widespread	O
among	O
the	O
bacteria	O
;	O
90	O
%	O
of	O
the	O
bacterial	O
genomes	O
in	O
the	O
Integrated	O
Microbial	O
Genomes	O
database	O
contain	O
a	O
gene	O
encoding	O
the	O
PTA_PTB	O
phosphotransacylase	O
(	O
Pfam	O
domain	O
PF01515	O
).	O
	O
The	O
primary	O
structure	O
of	O
PduL	O
homologs	O
is	O
subdivided	O
into	O
two	O
PF06130	O
domains	O
,	O
each	O
roughly	O
80	O
residues	O
in	O
length	O
.	O
	O
Structure	O
Determination	O
of	O
PduL	O
	O
Remarkably	O
,	O
after	O
removing	O
the	O
N	O
-	O
terminal	O
putative	O
EP	O
(	O
27	O
amino	O
acids	O
),	O
most	O
of	O
the	O
sPduLΔEP	B-mutant
protein	O
was	O
in	O
the	O
soluble	O
fraction	O
upon	O
cell	O
lysis	O
.	O
	O
A	O
CoA	O
cofactor	O
as	O
well	O
as	O
two	O
metal	O
ions	O
are	O
clearly	O
resolved	O
in	O
the	O
density	O
(	O
for	O
omit	O
maps	O
of	O
CoA	O
see	O
S2	O
Fig	O
).	O
	O
The	O
sequences	O
aligning	O
to	O
the	O
PF06130	O
domain	O
(	O
determined	O
by	O
BLAST	O
)	O
are	O
highlighted	O
in	O
red	O
and	O
blue	O
.	O
	O
Distances	O
between	O
atom	O
centers	O
are	O
indicated	O
in	O
Å	O
.	O
(	O
a	O
)	O
Coenzyme	O
A	O
containing	O
,	O
(	O
b	O
)	O
phosphate	O
-	O
bound	O
structure	O
.	O
	O
(	O
d	O
)–(	O
f	O
):	O
Chromatograms	O
of	O
sPduL	O
(	O
d	O
),	O
rPduL	O
(	O
e	O
),	O
and	O
pPduL	O
(	O
f	O
)	O
post	O
-	O
preparative	O
size	O
exclusion	O
chromatography	O
with	O
different	O
size	O
fractions	O
separated	O
,	O
applied	O
over	O
an	O
analytical	O
size	O
exclusion	O
column	O
(	O
see	O
Materials	O
and	O
Methods	O
).	O
	O
The	O
phosphate	O
contacts	O
both	O
zinc	O
atoms	O
(	O
Fig	O
4b	O
)	O
and	O
replaces	O
the	O
coordination	O
by	O
CoA	O
at	O
Zn1	O
;	O
the	O
coordination	O
for	O
Zn2	O
changes	O
from	O
octahedral	O
with	O
three	O
bound	O
waters	O
to	O
tetrahedral	O
with	O
a	O
phosphate	O
ion	O
as	O
one	O
of	O
the	O
ligands	O
(	O
Fig	O
4b	O
).	O
	O
The	O
two	O
zinc	O
atoms	O
are	O
slightly	O
closer	O
together	O
in	O
the	O
phosphate	O
-	O
bound	O
form	O
(	O
5	O
.	O
8	O
Å	O
vs	O
6	O
.	O
3	O
Å	O
),	O
possibly	O
due	O
to	O
the	O
bridging	O
effect	O
of	O
the	O
phosphate	O
.	O
	O
rPduL	O
full	O
length	O
runs	O
as	O
Mw	O
=	O
140	O
.	O
3	O
kDa	O
+/−	O
1	O
.	O
2	O
%	O
and	O
Mn	O
=	O
140	O
.	O
5	O
kDa	O
+/−	O
1	O
.	O
2	O
%.	O
	O
Moreover	O
,	O
the	O
PduL	O
crystal	O
structures	O
offer	O
a	O
clue	O
as	O
to	O
how	O
required	O
cofactors	O
enter	O
the	O
BMC	O
lumen	O
during	O
assembly	O
.	O
	O
The	O
native	O
substrate	O
for	O
the	O
forward	O
reaction	O
of	O
rPduL	O
and	O
pPduL	O
,	O
propionyl	O
-	O
CoA	O
,	O
most	O
likely	O
binds	O
to	O
the	O
enzyme	O
in	O
the	O
same	O
way	O
at	O
the	O
observed	O
nucleotide	O
and	O
pantothenic	O
acid	O
moiety	O
,	O
but	O
the	O
propionyl	O
group	O
in	O
the	O
CoA	O
-	O
thioester	O
might	O
point	O
in	O
a	O
different	O
direction	O
.	O
	O
Indeed	O
,	O
in	O
the	O
majority	O
of	O
PduLs	O
encoded	O
in	O
pvm	O
loci	O
,	O
Gln77	O
is	O
substituted	O
by	O
either	O
a	O
Tyr	O
or	O
Phe	O
,	O
whereas	O
it	O
is	O
typically	O
a	O
Gln	O
or	O
Glu	O
in	O
PduLs	O
in	O
all	O
other	O
BMC	O
types	O
that	O
degrade	O
acetyl	O
-	O
or	O
propionyl	O
-	O
CoA	O
.	O
A	O
comparison	O
of	O
the	O
PduL	O
active	O
site	O
to	O
that	O
of	O
the	O
functionally	O
identical	O
Pta	O
suggests	O
that	O
the	O
two	O
enzymes	O
have	O
distinctly	O
different	O
mechanisms	O
.	O
	O
The	O
two	O
high	O
-	O
resolution	O
crystal	O
structures	O
presented	O
here	O
will	O
serve	O
as	O
the	O
foundation	O
for	O
mechanistic	O
studies	O
on	O
this	O
noncanonical	O
PTAC	O
enzyme	O
to	O
determine	O
how	O
the	O
dimetal	O
active	O
site	O
functions	O
to	O
catalyze	O
both	O
forward	O
and	O
reverse	O
reactions	O
.	O
	O
There	O
could	O
be	O
some	O
intrinsic	O
biochemical	O
difference	O
between	O
the	O
two	O
enzymes	O
that	O
renders	O
PduL	O
a	O
more	O
attractive	O
candidate	O
for	O
encapsulation	O
in	O
a	O
BMC	O
—	O
for	O
example	O
,	O
PduL	O
might	O
be	O
more	O
amenable	O
to	O
tight	O
packaging	O
,	O
or	O
is	O
better	O
suited	O
for	O
the	O
chemical	O
microenvironment	O
formed	O
within	O
the	O
lumen	O
of	O
the	O
BMC	O
,	O
which	O
can	O
be	O
quite	O
different	O
from	O
the	O
cytosol	O
.	O
	O
A	O
detailed	O
understanding	O
of	O
the	O
underlying	O
principles	O
governing	O
the	O
assembly	O
and	O
internal	O
structural	O
organization	O
of	O
BMCs	O
is	O
a	O
requisite	O
for	O
synthetic	O
biologists	O
to	O
design	O
custom	O
nanoreactors	O
that	O
use	O
BMC	O
architectures	O
as	O
a	O
template	O
.	O
	O
Furthermore	O
,	O
given	O
the	O
growing	O
number	O
of	O
metabolosomes	O
implicated	O
in	O
pathogenesis	O
,	O
the	O
PduL	O
structure	O
will	O
be	O
useful	O
in	O
the	O
development	O
of	O
therapeutics	O
.	O
	O
EctC	O
forms	O
a	O
dimer	O
with	O
a	O
head	O
-	O
to	O
-	O
tail	O
arrangement	O
,	O
both	O
in	O
solution	O
and	O
in	O
the	O
crystal	O
structure	O
.	O
	O
We	O
show	O
for	O
the	O
first	O
time	O
that	O
ectoine	O
synthase	O
harbors	O
a	O
catalytically	O
important	O
metal	O
co	O
-	O
factor	O
;	O
metal	O
depletion	O
and	O
reconstitution	O
experiments	O
suggest	O
that	O
EctC	O
is	O
probably	O
an	O
iron	O
-	O
dependent	O
enzyme	O
.	O
	O
Structure	O
-	O
guided	O
site	O
-	O
directed	O
mutagenesis	O
experiments	O
targeting	O
amino	O
acid	O
residues	O
that	O
are	O
evolutionarily	O
highly	O
conserved	O
among	O
the	O
extended	O
EctC	O
protein	O
family	O
,	O
including	O
those	O
forming	O
the	O
presumptive	O
iron	O
-	O
binding	O
site	O
,	O
were	O
conducted	O
to	O
functionally	O
analyze	O
the	O
properties	O
of	O
the	O
resulting	O
EctC	O
variants	O
.	O
	O
This	O
stereospecific	O
chemical	O
modification	O
of	O
ectoine	O
(	O
Fig	O
1	O
)	O
is	O
catalyzed	O
by	O
the	O
ectoine	O
hydroxylase	O
(	O
EctD	O
)	O
(	O
EC	O
1	O
.	O
14	O
.	O
11	O
),	O
a	O
member	O
of	O
the	O
non	O
-	O
heme	O
containing	O
iron	O
(	O
II	O
)	O
and	O
2	O
-	O
oxoglutarate	O
-	O
dependent	O
dioxygenase	O
superfamily	O
.	O
	O
Scheme	O
of	O
the	O
ectoine	O
and	O
5	O
-	O
hydroxyectoine	O
biosynthetic	O
pathway	O
.	O
	O
The	O
EctC	O
protein	O
forms	O
a	O
dimer	O
in	O
solution	O
and	O
our	O
structural	O
analysis	O
identifies	O
it	O
as	O
a	O
member	O
of	O
the	O
cupin	O
superfamily	O
.	O
	O
(	O
Sa	O
)	O
EctC	O
is	O
a	O
highly	O
salt	O
-	O
tolerant	O
enzyme	O
since	O
it	O
exhibited	O
substantial	O
enzyme	O
activity	O
even	O
at	O
NaCl	O
and	O
KCl	O
concentrations	O
of	O
1	O
M	O
in	O
the	O
assay	O
buffer	O
(	O
S3c	O
and	O
S3d	O
Fig	O
).	O
	O
The	O
ectoine	O
synthase	O
is	O
a	O
metal	O
-	O
containing	O
protein	O
	O
The	O
amino	O
acid	O
sequences	O
of	O
20	O
selected	O
EctC	O
-	O
type	O
proteins	O
are	O
compared	O
.	O
	O
A	O
metal	O
cofactor	O
is	O
important	O
for	O
the	O
catalytic	O
activity	O
of	O
EctC	O
	O
To	O
address	O
these	O
questions	O
,	O
we	O
incubated	O
the	O
(	O
Sa	O
)	O
EctC	O
enzyme	O
with	O
increasing	O
concentrations	O
of	O
the	O
metal	O
chelator	O
ethylene	O
-	O
diamine	O
-	O
tetraacetic	O
-	O
acid	O
(	O
EDTA	O
)	O
and	O
subsequently	O
assayed	O
ectoine	O
synthase	O
activity	O
.	O
	O
The	O
EctC	O
-	O
catalyzed	O
ring	O
-	O
closure	O
of	O
N	O
-	O
γ	O
-	O
ADABA	O
to	O
form	O
ectoine	O
exhibited	O
Michaelis	O
-	O
Menten	O
-	O
kinetics	O
with	O
an	O
apparent	O
Km	O
of	O
4	O
.	O
9	O
±	O
0	O
.	O
5	O
mM	O
,	O
a	O
vmax	O
of	O
25	O
.	O
0	O
±	O
0	O
.	O
8	O
U	O
/	O
mg	O
and	O
a	O
kcat	O
of	O
7	O
.	O
2	O
s	O
-	O
1	O
(	O
S4a	O
Fig	O
).	O
	O
(	O
Sa	O
)	O
EctC	O
catalyzed	O
this	O
reaction	O
with	O
Michaelis	O
-	O
Menten	O
-	O
kinetics	O
exhibiting	O
an	O
apparent	O
Km	O
of	O
25	O
.	O
4	O
±	O
2	O
.	O
9	O
mM	O
,	O
a	O
vmax	O
of	O
24	O
.	O
6	O
±	O
1	O
.	O
0	O
U	O
/	O
mg	O
and	O
a	O
kcat	O
0	O
.	O
6	O
s	O
-	O
1	O
(	O
S4b	O
Fig	O
).	O
	O
However	O
,	O
two	O
crystal	O
forms	O
of	O
the	O
(	O
Sa	O
)	O
EctC	O
protein	O
in	O
the	O
absence	O
of	O
the	O
substrate	O
were	O
obtained	O
.	O
	O
Overall	O
fold	O
of	O
the	O
(	O
Sa	O
)	O
EctC	O
protein	O
	O
The	O
β	O
-	O
strands	O
are	O
numbered	O
β1	O
-	O
β11	O
and	O
the	O
helices	O
α	O
-	O
I	O
to	O
α	O
-	O
II	O
.	O
	O
The	O
entrance	O
to	O
the	O
active	O
site	O
of	O
the	O
ectoine	O
synthase	O
is	O
marked	O
.	O
	O
(	O
c	O
)	O
Overlay	O
of	O
the	O
“	O
semi	O
-	O
closed	O
”	O
and	O
“	O
open	O
”	O
(	O
Sa	O
)	O
EctC	O
structures	O
.	O
	O
Hence	O
,	O
(	O
Sa	O
)	O
EctC	O
adopts	O
an	O
overall	O
bowl	O
shape	O
in	O
which	O
one	O
side	O
is	O
opened	O
towards	O
the	O
solvent	O
(	O
Fig	O
4a	O
to	O
4c	O
).	O
	O
The	O
formation	O
of	O
this	O
α	O
-	O
II	O
helix	O
induces	O
a	O
reorientation	O
and	O
shift	O
of	O
a	O
long	O
unstructured	O
loop	O
(	O
as	O
observed	O
in	O
the	O
“	O
open	O
”	O
structure	O
)	O
connecting	O
β4	O
and	O
β6	O
,	O
resulting	O
in	O
the	O
formation	O
of	O
the	O
stable	O
β	O
-	O
strand	O
β5	O
as	O
observed	O
in	O
the	O
“	O
semi	O
-	O
closed	O
”	O
state	O
of	O
the	O
(	O
Sa	O
)	O
EctC	O
protein	O
(	O
Fig	O
4a	O
).	O
	O
Both	O
the	O
SEC	O
analysis	O
and	O
the	O
HPLC	O
-	O
MALS	O
experiments	O
(	O
S2b	O
Fig	O
)	O
have	O
shown	O
that	O
the	O
ectoine	O
synthase	O
from	O
S	O
.	O
alaskensis	O
is	O
a	O
dimer	O
in	O
solution	O
.	O
	O
The	O
crystal	O
structure	O
of	O
this	O
protein	O
reflects	O
this	O
quaternary	O
arrangement	O
.	O
	O
As	O
calculated	O
with	O
PDBePISA	O
,	O
the	O
surface	O
area	O
buried	O
upon	O
dimer	O
formation	O
is	O
1462	O
Å2	O
,	O
which	O
is	O
20	O
.	O
5	O
%	O
of	O
the	O
total	O
accessible	O
surface	O
of	O
a	O
monomer	O
of	O
this	O
protein	O
.	O
	O
In	O
the	O
“	O
open	O
”	O
(	O
Sa	O
)	O
EctC	O
structure	O
,	O
one	O
monomer	O
is	O
present	O
in	O
the	O
asymmetric	O
unit	O
.	O
	O
We	O
therefore	O
inspected	O
the	O
crystal	O
packing	O
and	O
analyzed	O
the	O
monomer	O
-	O
monomer	O
interactions	O
with	O
symmetry	O
related	O
molecules	O
to	O
elucidate	O
whether	O
a	O
physiologically	O
relevant	O
dimer	O
could	O
be	O
deduced	O
from	O
this	O
crystal	O
form	O
as	O
well	O
.	O
	O
These	O
additional	O
amino	O
acids	O
fold	O
into	O
a	O
small	O
helix	O
,	O
which	O
seals	O
the	O
open	O
cavity	O
of	O
the	O
cupin	O
-	O
fold	O
of	O
the	O
(	O
Sa	O
)	O
EctC	O
protein	O
(	O
Fig	O
4a	O
).	O
	O
As	O
a	O
result	O
,	O
the	O
newly	O
formed	O
β	O
-	O
strand	O
β5	O
is	O
reoriented	O
and	O
moved	O
by	O
2	O
.	O
4	O
Å	O
within	O
the	O
“	O
semi	O
-	O
closed	O
”	O
(	O
Sa	O
)	O
EctC	O
structure	O
(	O
Fig	O
4a	O
to	O
4c	O
).	O
	O
Therefore	O
the	O
sealing	O
of	O
the	O
cupin	O
fold	O
,	O
as	O
described	O
above	O
,	O
seem	O
to	O
have	O
an	O
indirect	O
influence	O
on	O
the	O
architecture	O
of	O
the	O
postulated	O
iron	O
-	O
binding	O
site	O
.	O
	O
In	O
the	O
“	O
open	O
”	O
structure	O
of	O
the	O
(	O
Sa	O
)	O
EctC	O
protein	O
,	O
this	O
interaction	O
does	O
not	O
occur	O
since	O
Glu	O
-	O
115	O
is	O
rotated	O
outwards	O
(	O
Fig	O
6a	O
and	O
6b	O
).	O
	O
Hence	O
,	O
one	O
might	O
speculate	O
that	O
this	O
missing	O
interaction	O
might	O
be	O
responsible	O
for	O
the	O
flexibility	O
of	O
the	O
carboxy	O
-	O
terminus	O
in	O
the	O
“	O
open	O
”	O
(	O
Sa	O
)	O
EctC	O
structure	O
and	O
consequently	O
results	O
in	O
less	O
well	O
defined	O
electron	O
density	O
in	O
this	O
region	O
.	O
	O
These	O
distances	O
are	O
to	O
long	O
when	O
compared	O
to	O
other	O
iron	O
binding	O
sites	O
,	O
a	O
fact	O
that	O
might	O
be	O
caused	O
by	O
the	O
absence	O
of	O
the	O
proper	O
substrate	O
in	O
the	O
(	O
Sa	O
)	O
EctC	O
crystal	O
structure	O
.	O
	O
Since	O
both	O
the	O
refinement	O
and	O
the	O
distance	O
did	O
not	O
clearly	O
identify	O
an	O
iron	O
molecule	O
,	O
we	O
decided	O
to	O
conservatively	O
place	O
a	O
water	O
molecule	O
at	O
this	O
position	O
.	O
	O
Only	O
His	O
-	O
93	O
is	O
slightly	O
rotated	O
inwards	O
in	O
the	O
“	O
semi	O
-	O
closed	O
”	O
structure	O
,	O
most	O
likely	O
due	O
to	O
formation	O
of	O
β	O
-	O
strand	O
β5	O
as	O
described	O
above	O
.	O
	O
Taken	O
together	O
,	O
this	O
observations	O
indicate	O
,	O
that	O
the	O
architecture	O
of	O
the	O
presumptive	O
iron	O
-	O
binding	O
site	O
is	O
pre	O
-	O
set	O
for	O
the	O
binding	O
of	O
the	O
catalytically	O
important	O
metal	O
by	O
the	O
ectoine	O
synthase	O
.	O
	O
This	O
is	O
in	O
contrast	O
to	O
the	O
high	O
-	O
resolution	O
“	O
open	O
”	O
structure	O
of	O
the	O
(	O
Sa	O
)	O
EctC	O
protein	O
where	O
no	O
additional	O
electron	O
density	O
was	O
observed	O
after	O
refinement	O
.	O
	O
When	O
analyzing	O
the	O
interactions	O
of	O
this	O
compound	O
within	O
the	O
(	O
Sa	O
)	O
EctC	O
protein	O
,	O
we	O
found	O
that	O
it	O
is	O
bound	O
via	O
interactions	O
with	O
Trp	O
-	O
21	O
and	O
Ser	O
-	O
23	O
of	O
β	O
-	O
sheet	O
β3	O
,	O
Thr	O
-	O
40	O
located	O
in	O
β	O
-	O
sheet	O
β4	O
,	O
and	O
Cys	O
-	O
105	O
and	O
Phe	O
-	O
107	O
,	O
which	O
are	O
both	O
part	O
of	O
β	O
-	O
sheet	O
β11	O
.	O
	O
As	O
described	O
above	O
,	O
the	O
side	O
chains	O
of	O
Glu	O
-	O
57	O
,	O
Tyr	O
-	O
85	O
,	O
and	O
His	O
-	O
93	O
are	O
probably	O
involved	O
in	O
iron	O
binding	O
(	O
Table	O
1	O
and	O
Fig	O
6a	O
).	O
	O
However	O
,	O
the	O
Cys	B-mutant
-	I-mutant
105	I-mutant
/	I-mutant
Ala	I-mutant
variant	O
was	O
practically	O
catalytically	O
inactive	O
while	O
largely	O
maintaining	O
its	O
iron	O
content	O
(	O
Table	O
1	O
).	O
	O
We	O
observed	O
two	O
amino	O
acid	O
substitutions	O
that	O
simultaneously	O
strongly	O
affected	O
enzyme	O
activity	O
and	O
iron	O
content	O
;	O
these	O
were	O
the	O
Tyr	B-mutant
-	I-mutant
52	I-mutant
/	I-mutant
Ala	I-mutant
and	O
the	O
His	B-mutant
-	I-mutant
55	I-mutant
/	I-mutant
Ala	I-mutant
(	O
Sa	O
)	O
EctC	O
protein	O
variants	O
(	O
Table	O
1	O
).	O
	O
The	O
carboxy	O
-	O
terminal	O
region	O
of	O
the	O
(	O
Sa	O
)	O
EctC	O
protein	O
is	O
held	O
in	O
its	O
position	O
via	O
an	O
interaction	O
of	O
Glu	O
-	O
115	O
with	O
His	O
-	O
55	O
,	O
where	O
His	O
-	O
55	O
in	O
turn	O
interacts	O
with	O
Pro	O
-	O
110	O
(	O
Fig	O
6a	O
and	O
6b	O
).	O
	O
The	O
Glu	B-mutant
-	I-mutant
115	I-mutant
/	I-mutant
Ala	I-mutant
mutant	O
possessed	O
wild	O
-	O
type	O
levels	O
of	O
iron	O
,	O
whereas	O
the	O
iron	O
content	O
of	O
the	O
His	B-mutant
-	I-mutant
55	I-mutant
/	I-mutant
Ala	I-mutant
substitutions	O
dropped	O
to	O
15	O
%	O
of	O
the	O
wild	O
-	O
type	O
level	O
(	O
Table	O
1	O
).	O
	O
As	O
a	O
consequence	O
of	O
the	O
structural	O
relatedness	O
of	O
EctC	O
and	O
RemF	O
and	O
the	O
type	O
of	O
chemical	O
reaction	O
these	O
two	O
enzymes	O
catalyze	O
,	O
is	O
now	O
understandable	O
why	O
bona	O
fide	O
EctC	O
-	O
type	O
proteins	O
are	O
frequently	O
(	O
mis	O
)-	O
annotated	O
in	O
microbial	O
genome	O
sequences	O
as	O
“	O
RemF	O
-	O
like	O
”	O
proteins	O
.	O
	O
Except	O
for	O
some	O
cupin	O
-	O
related	O
proteins	O
that	O
seem	O
to	O
function	O
as	O
metallo	O
-	O
chaperones	O
,	O
the	O
bound	O
metal	O
is	O
typically	O
an	O
essential	O
part	O
of	O
the	O
active	O
sites	O
.	O
	O
The	O
architecture	O
of	O
the	O
metal	O
center	O
of	O
ectoine	O
synthase	O
seems	O
to	O
be	O
subjected	O
to	O
considerable	O
evolutionary	O
constraints	O
.	O
	O
This	O
set	O
of	O
data	O
and	O
the	O
fact	O
that	O
the	O
targeted	O
residues	O
are	O
strongly	O
conserved	O
among	O
EctC	O
-	O
type	O
proteins	O
(	O
Fig	O
2	O
)	O
is	O
consistent	O
with	O
their	O
potential	O
role	O
in	O
N	O
-	O
γ	O
-	O
ADABA	O
binding	O
or	O
enzyme	O
catalysis	O
.	O
	O
Because	O
microbial	O
ectoine	O
producers	O
can	O
colonize	O
ecological	O
niches	O
with	O
rather	O
different	O
physicochemical	O
attributes	O
,	O
it	O
seems	O
promising	O
to	O
exploit	O
this	O
considerable	O
biodiversity	O
to	O
identify	O
EctC	O
proteins	O
with	O
enhanced	O
protein	O
stability	O
.	O
	O
Structural	O
basis	O
for	O
the	O
regulation	O
of	O
enzymatic	O
activity	O
of	O
Regnase	O
-	O
1	O
by	O
domain	O
-	O
domain	O
interactions	O
	O
Domain	O
structures	O
of	O
Regnase	O
-	O
1	O
	O
The	O
domain	O
structures	O
of	O
NTD	O
,	O
ZF	O
,	O
and	O
CTD	O
were	O
determined	O
by	O
NMR	O
(	O
Fig	O
.	O
1b	O
,	O
d	O
,	O
e	O
).	O
	O
Based	O
on	O
the	O
decrease	O
in	O
the	O
free	O
RNA	O
fluorescence	O
band	O
,	O
we	O
evaluated	O
the	O
contribution	O
of	O
each	O
domain	O
of	O
Regnase	O
-	O
1	O
to	O
RNA	O
binding	O
.	O
	O
Contribution	O
of	O
each	O
domain	O
of	O
Regnase	O
-	O
1	O
to	O
RNase	O
activity	O
	O
It	O
should	O
be	O
noted	O
that	O
NTD	B-mutant
-	I-mutant
PIN	I-mutant
(	I-mutant
DDNN	I-mutant
)-	I-mutant
ZF	I-mutant
,	O
which	O
possesses	O
the	O
NTD	O
but	O
lacks	O
the	O
catalytic	O
residues	O
in	O
PIN	O
,	O
completely	O
lost	O
all	O
RNase	O
activity	O
(	O
Fig	O
.	O
1g	O
,	O
right	O
panel	O
),	O
as	O
expected	O
,	O
confirming	O
that	O
the	O
RNase	O
catalytic	O
center	O
is	O
located	O
in	O
the	O
PIN	O
domain	O
.	O
	O
By	O
comparison	O
with	O
the	O
elution	O
volume	O
of	O
standard	O
marker	O
proteins	O
,	O
the	O
PIN	O
domain	O
was	O
assumed	O
to	O
be	O
in	O
equilibrium	O
between	O
a	O
monomer	O
and	O
a	O
dimer	O
in	O
solution	O
at	O
concentrations	O
in	O
the	O
20	O
–	O
200	O
μM	O
range	O
.	O
	O
The	O
crystal	O
structure	O
of	O
the	O
PIN	O
domain	O
has	O
been	O
determined	O
in	O
three	O
distinct	O
crystal	O
forms	O
with	O
a	O
space	O
group	O
of	O
P3121	O
(	O
form	O
I	O
in	O
this	O
study	O
and	O
PDB	O
ID	O
3V33	O
),	O
P3221	O
(	O
form	O
II	O
in	O
this	O
study	O
),	O
and	O
P41	O
(	O
PDB	O
ID	O
3V32	O
and	O
3V34	O
),	O
respectively	O
.	O
	O
Mutation	O
of	O
Arg215	O
,	O
whose	O
side	O
chain	O
faces	O
to	O
the	O
opposite	O
side	O
of	O
the	O
oligomeric	O
surface	O
,	O
to	O
Glu	O
preserved	O
the	O
monomer	O
/	O
dimer	O
equilibrium	O
,	O
similar	O
to	O
the	O
wild	O
type	O
.	O
	O
Therefore	O
,	O
we	O
concluded	O
that	O
head	O
-	O
to	O
-	O
tail	O
PIN	O
dimerization	O
,	O
together	O
with	O
the	O
NTD	O
,	O
are	O
required	O
for	O
Regnase	O
-	O
1	O
RNase	O
activity	O
in	O
vitro	O
.	O
	O
Likewise	O
,	O
upon	O
addition	O
of	O
the	O
PIN	O
domain	O
,	O
NMR	O
signals	O
derived	O
from	O
R56	O
,	O
L58	O
-	O
G59	O
,	O
and	O
V86	O
-	O
H88	O
in	O
the	O
NTD	O
exhibited	O
large	O
chemical	O
shift	O
changes	O
and	O
residues	O
D53	O
,	O
F55	O
,	O
K57	O
,	O
Y60	O
-	O
S61	O
,	O
V68	O
,	O
T80	O
-	O
G83	O
,	O
L85	O
,	O
and	O
G89	O
of	O
the	O
NTD	O
as	O
well	O
as	O
side	O
chain	O
amide	O
signals	O
of	O
N79	O
exhibited	O
small	O
but	O
appreciable	O
chemical	O
shift	O
changes	O
(	O
Fig	O
.	O
3b	O
and	O
Supplementary	O
Fig	O
.	O
5	O
).	O
	O
The	O
importance	O
of	O
residues	O
W182	O
and	O
R183	O
can	O
readily	O
be	O
understood	O
in	O
terms	O
of	O
the	O
monomeric	O
PIN	O
structure	O
as	O
they	O
are	O
located	O
near	O
to	O
the	O
RNase	O
catalytic	O
site	O
;	O
however	O
,	O
the	O
importance	O
of	O
residue	O
K184	O
,	O
which	O
points	O
away	O
from	O
the	O
active	O
site	O
is	O
more	O
easily	O
rationalized	O
in	O
terms	O
of	O
the	O
oligomeric	O
structure	O
,	O
in	O
which	O
the	O
“	O
secondary	O
”	O
chain	O
’	O
s	O
residue	O
K184	O
is	O
positioned	O
near	O
the	O
“	O
primary	O
”	O
chain	O
’	O
s	O
catalytic	O
site	O
(	O
Fig	O
.	O
4	O
).	O
	O
It	O
should	O
be	O
noted	O
that	O
the	O
putative	O
-	O
RNA	O
binding	O
residues	O
K184	O
and	O
R214	O
are	O
unique	O
to	O
Regnase	O
-	O
1	O
among	O
PIN	O
domains	O
.	O
	O
Molecular	O
mechanism	O
of	O
target	O
mRNA	O
cleavage	O
by	O
the	O
PIN	O
dimer	O
	O
Our	O
mutational	O
experiments	O
indicated	O
that	O
the	O
observed	O
dimer	O
is	O
functional	O
and	O
that	O
the	O
role	O
of	O
the	O
secondary	O
PIN	O
domain	O
is	O
to	O
position	O
Regnase	O
-	O
1	O
-	O
unique	O
RNA	O
binding	O
residues	O
near	O
the	O
active	O
site	O
of	O
the	O
primary	O
PIN	O
domain	O
.	O
	O
We	O
determined	O
the	O
individual	O
domain	O
structures	O
of	O
Regnase	O
-	O
1	O
by	O
NMR	O
and	O
X	O
-	O
ray	O
crystallography	O
.	O
	O
Both	O
the	O
mouse	O
and	O
human	O
PIN	O
domains	O
form	O
head	O
-	O
to	O
-	O
tail	O
oligomers	O
in	O
three	O
distinct	O
crystal	O
forms	O
.	O
	O
In	O
contrast	O
,	O
our	O
gel	O
filtration	O
data	O
,	O
mutational	O
analyses	O
,	O
and	O
NMR	O
spectra	O
all	O
indicate	O
that	O
the	O
PIN	O
domain	O
forms	O
a	O
head	O
-	O
to	O
-	O
tail	O
dimer	O
in	O
solution	O
in	O
a	O
manner	O
similar	O
to	O
the	O
crystal	O
structure	O
.	O
	O
Taken	O
together	O
,	O
this	O
suggests	O
that	O
the	O
NTD	O
and	O
the	O
PIN	O
domain	O
compete	O
for	O
a	O
common	O
binding	O
site	O
.	O
	O
While	O
further	O
investigations	O
on	O
the	O
domain	O
-	O
domain	O
interactions	O
of	O
Regnase	O
-	O
1	O
in	O
vivo	O
are	O
necessary	O
,	O
these	O
intramolecular	O
and	O
intermolecular	O
domain	O
interactions	O
of	O
Regnase	O
-	O
1	O
appear	O
to	O
structurally	O
constrain	O
Regnase	O
-	O
1activity	O
,	O
which	O
,	O
in	O
turn	O
,	O
enables	O
tight	O
regulation	O
of	O
immune	O
responses	O
.	O
	O
The	O
percentage	O
of	O
the	O
bound	O
IL	O
-	O
6	O
was	O
calculated	O
based	O
on	O
the	O
fluorescence	O
intensities	O
of	O
the	O
free	O
IL	O
-	O
6	O
quantified	O
in	O
(	O
f	O
).	O
	O
(	O
b	O
)	O
Dimer	O
structure	O
of	O
the	O
PIN	O
domain	O
.	O
	O
Two	O
PIN	O
molecules	O
in	O
the	O
crystal	O
were	O
colored	O
white	O
and	O
green	O
,	O
respectively	O
.	O
	O
(	O
a	O
)	O
NMR	O
analyses	O
of	O
the	O
NTD	O
-	O
binding	O
to	O
the	O
PIN	O
domain	O
.	O
	O
The	O
residues	O
with	O
significant	O
chemical	O
shift	O
changes	O
were	O
labeled	O
in	O
the	O
overlaid	O
spectra	O
(	O
left	O
)	O
and	O
colored	O
red	O
on	O
the	O
surface	O
and	O
ribbon	O
structure	O
of	O
the	O
PIN	O
domain	O
(	O
right	O
).	O
	O
The	O
NTD	O
and	O
the	O
PIN	O
domain	O
are	O
shown	O
in	O
cyan	O
and	O
white	O
,	O
respectively	O
.	O
	O
Catalytic	O
residues	O
of	O
the	O
PIN	O
domain	O
are	O
shown	O
in	O
sticks	O
,	O
and	O
the	O
residues	O
that	O
exhibited	O
significant	O
chemical	O
shift	O
changes	O
in	O
(	O
a	O
,	O
b	O
)	O
were	O
labeled	O
.	O
	O
(	O
b	O
)	O
In	O
vitro	O
cleavage	O
assay	O
of	O
basic	O
residue	O
mutants	O
for	O
Regnase	O
-	O
1	O
mRNA	O
.	O
	O
The	O
mutations	O
whose	O
RNase	O
activities	O
were	O
not	O
increased	O
in	O
the	O
presence	O
of	O
DDNN	B-mutant
mutant	O
were	O
colored	O
in	O
blue	O
on	O
the	O
primary	O
PIN	O
.	O
	O
In	O
the	O
MEROPS	O
peptidase	O
database	O
,	O
clan	O
CD	O
contains	O
groups	O
(	O
or	O
families	O
)	O
of	O
cysteine	O
peptidases	O
that	O
share	O
some	O
highly	O
conserved	O
structural	O
elements	O
.	O
	O
The	O
structure	O
was	O
analyzed	O
,	O
and	O
the	O
enzyme	O
was	O
biochemically	O
characterized	O
to	O
provide	O
the	O
first	O
structure	O
/	O
function	O
correlation	O
for	O
a	O
C11	O
peptidase	O
.	O
	O
The	O
crystal	O
structure	O
of	O
the	O
catalytically	O
active	O
form	O
of	O
PmC11	O
revealed	O
an	O
extended	O
caspase	O
-	O
like	O
α	O
/	O
β	O
/	O
α	O
sandwich	O
architecture	O
comprised	O
of	O
a	O
central	O
nine	O
-	O
stranded	O
β	O
-	O
sheet	O
,	O
with	O
an	O
unusual	O
C	O
-	O
terminal	O
domain	O
(	O
CTD	O
),	O
starting	O
at	O
Lys250	O
.	O
	O
The	O
structure	O
also	O
includes	O
two	O
short	O
β	O
-	O
hairpins	O
(	O
βA	O
–	O
βB	O
and	O
βD	O
–	O
βE	O
)	O
and	O
a	O
small	O
β	O
-	O
sheet	O
(	O
βC	O
–	O
βF	O
),	O
which	O
is	O
formed	O
from	O
two	O
distinct	O
regions	O
of	O
the	O
sequence	O
(	O
βC	O
precedes	O
α11	O
,	O
α12	O
and	O
β9	O
,	O
whereas	O
βF	O
follows	O
the	O
βD	O
-	O
βE	O
hairpin	O
)	O
in	O
the	O
middle	O
of	O
the	O
CTD	O
(	O
Fig	O
.	O
1B	O
).	O
	O
His133	O
and	O
Cys179	O
were	O
found	O
at	O
locations	O
structurally	O
homologous	O
to	O
the	O
caspase	O
catalytic	O
dyad	O
,	O
and	O
other	O
clan	O
CD	O
structures	O
,	O
at	O
the	O
C	O
termini	O
of	O
strands	O
β5	O
and	O
β6	O
,	O
respectively	O
(	O
Figs	O
.	O
1	O
,	O
A	O
and	O
B	O
,	O
and	O
2A	O
).	O
	O
A	O
multiple	O
sequence	O
alignment	O
of	O
C11	O
proteins	O
revealed	O
that	O
these	O
residues	O
are	O
highly	O
conserved	O
(	O
data	O
not	O
shown	O
).	O
	O
B	O
,	O
size	O
exclusion	O
chromatography	O
of	O
PmC11	O
.	O
	O
Incubation	O
of	O
PmC11	O
at	O
37	O
°	O
C	O
for	O
16	O
h	O
,	O
resulted	O
in	O
a	O
fully	O
processed	O
enzyme	O
that	O
remained	O
as	O
an	O
intact	O
monomer	O
when	O
applied	O
to	O
a	O
size	O
-	O
exclusion	O
column	O
(	O
Fig	O
.	O
2B	O
).	O
	O
As	O
expected	O
,	O
PmC11	O
showed	O
no	O
activity	O
against	O
substrates	O
with	O
Pro	O
or	O
Asp	O
in	O
P1	O
but	O
was	O
active	O
toward	O
substrates	O
with	O
a	O
basic	O
residue	O
in	O
P1	O
such	O
as	O
Bz	O
-	O
R	O
-	O
AMC	O
,	O
Z	O
-	O
GGR	O
-	O
AMC	O
,	O
and	O
BOC	O
-	O
VLK	O
-	O
AMC	O
.	O
	O
The	O
rate	O
of	O
cleavage	O
was	O
∼	O
3	O
-	O
fold	O
greater	O
toward	O
the	O
single	O
Arg	O
substrate	O
Bz	O
-	O
R	O
-	O
AMC	O
than	O
for	O
the	O
other	O
two	O
(	O
Fig	O
.	O
2F	O
)	O
and	O
,	O
unexpectedly	O
,	O
PmC11	O
showed	O
no	O
activity	O
toward	O
BOC	O
-	O
K	O
-	O
AMC	O
.	O
	O
These	O
results	O
confirm	O
that	O
PmC11	O
accepts	O
substrates	O
containing	O
Arg	O
or	O
Lys	O
in	O
P1	O
with	O
a	O
possible	O
preference	O
for	O
Arg	O
.	O
	O
Because	O
PmC11	O
recognizes	O
basic	O
substrates	O
,	O
the	O
tetrapeptide	O
inhibitor	O
Z	O
-	O
VRPR	O
-	O
FMK	O
was	O
tested	O
as	O
an	O
enzyme	O
inhibitor	O
and	O
was	O
found	O
to	O
inhibit	O
both	O
the	O
autoprocessing	O
and	O
activity	O
of	O
PmC11	O
(	O
Fig	O
.	O
3A	O
).	O
	O
In	O
the	O
structure	O
of	O
PmC11	O
,	O
Asp207	O
resides	O
on	O
a	O
flexible	O
loop	O
pointing	O
away	O
from	O
the	O
S1	O
binding	O
pocket	O
(	O
Fig	O
.	O
3C	O
).	O
	O
The	O
position	O
and	O
orientation	O
of	O
Z	O
-	O
VRPR	O
-	O
FMK	O
was	O
taken	O
from	O
superposition	O
of	O
the	O
PmC11	O
and	O
MALTI_P	O
structures	O
and	O
indicates	O
the	O
presumed	O
active	O
site	O
of	O
PmC11	O
.	O
	O
C	O
,	O
divalent	O
cations	O
do	O
not	O
increase	O
the	O
activity	O
of	O
PmC11	O
.	O
	O
The	O
cleavage	O
of	O
Bz	O
-	O
R	O
-	O
AMC	O
by	O
PmC11	O
was	O
measured	O
in	O
the	O
presence	O
of	O
the	O
cations	O
Ca2	O
+,	O
Mn2	O
+,	O
Zn2	O
+,	O
Co2	O
+,	O
Cu2	O
+,	O
Mg2	O
+,	O
and	O
Fe3	O
+	O
with	O
EGTA	O
as	O
a	O
negative	O
control	O
,	O
and	O
relative	O
fluorescence	O
measured	O
against	O
time	O
(	O
min	O
).	O
	O
The	O
addition	O
of	O
cations	O
produced	O
no	O
improvement	O
in	O
activity	O
of	O
PmC11	O
when	O
compared	O
in	O
the	O
presence	O
of	O
EGTA	O
,	O
suggesting	O
that	O
PmC11	O
does	O
not	O
require	O
metal	O
ions	O
for	O
proteolytic	O
activity	O
.	O
	O
Several	O
other	O
members	O
of	O
clan	O
CD	O
require	O
processing	O
for	O
full	O
activation	O
including	O
legumain	O
,	O
gingipain	O
-	O
R	O
,	O
MARTX	O
-	O
CPD	O
,	O
and	O
the	O
effector	O
caspases	O
,	O
e	O
.	O
g	O
.	O
caspase	O
-	O
7	O
.	O
	O
The	O
caspases	O
and	O
gingipain	O
-	O
R	O
both	O
undergo	O
intermolecular	O
(	O
trans	O
)	O
cleavage	O
and	O
legumain	O
and	O
MARTX	O
-	O
CPD	O
are	O
reported	O
to	O
perform	O
intramolecular	O
(	O
cis	O
)	O
cleavage	O
.	O
	O
The	O
PmC11	O
structure	O
should	O
provide	O
a	O
good	O
basis	O
for	O
structural	O
modeling	O
and	O
,	O
given	O
the	O
importance	O
of	O
other	O
clan	O
CD	O
enzymes	O
,	O
this	O
work	O
should	O
also	O
advance	O
the	O
exploration	O
of	O
these	O
peptidases	O
and	O
potentially	O
identify	O
new	O
biologically	O
important	O
substrates	O
.	O
	O
The	O
chemically	O
most	O
complex	O
modification	O
in	O
eukaryotic	O
rRNA	O
is	O
the	O
conserved	O
hypermodified	O
nucleotide	O
N1	O
-	O
methyl	O
-	O
N3	O
-	O
aminocarboxypropyl	O
-	O
pseudouridine	O
(	O
m1acp3Ψ	O
)	O
located	O
next	O
to	O
the	O
P	O
-	O
site	O
tRNA	O
on	O
the	O
small	O
subunit	O
18S	O
rRNA	O
.	O
	O
While	O
S	O
-	O
adenosylmethionine	O
was	O
identified	O
as	O
the	O
source	O
of	O
the	O
aminocarboxypropyl	O
(	O
acp	O
)	O
group	O
more	O
than	O
40	O
years	O
ago	O
the	O
enzyme	O
catalyzing	O
the	O
acp	O
transfer	O
remained	O
elusive	O
.	O
	O
In	O
Saccharomyces	O
cerevisiae	O
18S	O
rRNA	O
contains	O
four	O
base	O
methylations	O
,	O
two	O
acetylations	O
and	O
a	O
single	O
3	O
-	O
amino	O
-	O
3	O
-	O
carboxypropyl	O
(	O
acp	O
)	O
modification	O
,	O
whereas	O
six	O
base	O
methylations	O
are	O
present	O
in	O
the	O
25S	O
rRNA	O
.	O
	O
While	O
in	O
humans	O
the	O
18S	O
rRNA	O
base	O
modifications	O
are	O
highly	O
conserved	O
,	O
only	O
three	O
of	O
the	O
yeast	O
base	O
modifications	O
catalyzed	O
by	O
ScRrp8	O
/	O
HsNML	O
,	O
ScRcm1	O
/	O
HsNSUN5	O
and	O
ScNop2	O
/	O
HsNSUN1	O
are	O
preserved	O
in	O
the	O
corresponding	O
human	O
28S	O
rRNA	O
.	O
	O
They	O
might	O
contribute	O
to	O
increased	O
RNA	O
stability	O
by	O
providing	O
additional	O
hydrogen	O
bonds	O
(	O
pseudouridines	O
),	O
improved	O
base	O
stacking	O
(	O
pseudouridines	O
and	O
base	O
methylations	O
)	O
or	O
an	O
increased	O
resistance	O
against	O
hydrolysis	O
(	O
ribose	O
methylations	O
).	O
	O
Defects	O
of	O
rRNA	O
modification	O
enzymes	O
often	O
lead	O
to	O
disturbed	O
ribosome	O
biogenesis	O
or	O
functionally	O
impaired	O
ribosomes	O
,	O
although	O
the	O
lack	O
of	O
individual	O
rRNA	O
modifications	O
often	O
has	O
no	O
or	O
only	O
a	O
slight	O
influence	O
on	O
the	O
cell	O
.	O
	O
The	O
asterisk	O
indicates	O
the	O
C1	O
-	O
atom	O
labeled	O
in	O
the	O
14C	O
-	O
incorporation	O
assay	O
.	O
	O
(	O
C	O
)	O
14C	O
-	O
acp	O
labeling	O
of	O
18S	O
rRNAs	O
.	O
	O
The	O
primer	O
extension	O
arrest	O
is	O
reduced	O
in	O
HTC116	O
cells	O
transfected	O
with	O
siRNAs	O
544	O
and	O
545	O
.	O
	O
As	O
previously	O
reported	O
this	O
shoulder	O
was	O
identified	O
by	O
ESI	O
-	O
MS	O
as	O
corresponding	O
to	O
m1acp3Ψ	O
.	O
	O
Whereas	O
the	O
acp	O
labeling	O
of	O
18S	O
rRNA	O
was	O
clearly	O
present	O
in	O
the	O
wild	O
type	O
strain	O
no	O
radioactive	O
labeling	O
could	O
be	O
observed	O
in	O
a	O
Δtsr3	B-mutant
strain	O
(	O
Figure	O
1C	O
).	O
	O
Human	O
18S	O
rRNA	O
has	O
also	O
been	O
shown	O
to	O
contain	O
m1acp3Ψ	O
in	O
the	O
18S	O
rRNA	O
at	O
position	O
1248	O
.	O
	O
By	O
comparison	O
,	O
treating	O
cells	O
with	O
siRNA	O
545	O
,	O
which	O
only	O
reduced	O
the	O
TSR3	O
mRNA	O
to	O
20	O
%,	O
did	O
not	O
markedly	O
reduced	O
the	O
acp	O
signal	O
.	O
	O
The	O
TSR3	O
gene	O
was	O
genetically	O
modified	O
at	O
its	O
native	O
locus	O
,	O
resulting	O
in	O
a	O
C	O
-	O
terminal	O
fusion	O
of	O
Tsr3	O
with	O
a	O
3xHA	O
epitope	O
expressed	O
by	O
the	O
native	O
promotor	O
in	O
yeast	O
strain	O
CEN	O
.	O
BM258	O
-	O
5B	O
.	O
	O
In	O
accordance	O
with	O
the	O
synthetic	O
sick	O
growth	O
phenotype	O
the	O
paromomycin	O
and	O
hygromycin	O
B	O
hypersensitivity	O
further	O
increased	O
in	O
a	O
Δtsr3	B-mutant
Δsnr35	I-mutant
recombination	O
strain	O
(	O
Figure	O
2B	O
).	O
	O
Domain	O
characterization	O
of	O
yeast	O
Tsr3	O
and	O
correlation	O
of	O
acp	O
modification	O
with	O
late	O
18S	O
rRNA	O
processing	O
steps	O
.	O
(	O
A	O
)	O
Scheme	O
of	O
the	O
TSR3	O
gene	O
with	O
truncation	O
positions	O
in	O
the	O
open	O
reading	O
frame	O
.	O
	O
The	O
loop	O
connecting	O
β2	O
and	O
β3	O
contains	O
a	O
single	O
turn	O
of	O
a	O
310	O
-	O
helix	O
.	O
Helices	O
α1	O
and	O
α2	O
are	O
located	O
on	O
one	O
side	O
of	O
the	O
five	O
-	O
stranded	O
β	O
-	O
sheet	O
while	O
α3	O
packs	O
against	O
the	O
opposite	O
β	O
-	O
sheet	O
surface	O
.	O
	O
The	O
bound	O
S	O
-	O
adenosylmethionine	O
is	O
shown	O
in	O
a	O
stick	O
representation	O
and	O
colored	O
by	O
atom	O
type	O
.	O
	O
The	O
color	O
coding	O
is	O
the	O
same	O
as	O
in	O
(	O
A	O
).	O
(	O
C	O
)	O
Structural	O
superposition	O
of	O
the	O
X	O
-	O
ray	O
structures	O
of	O
VdTsr3	O
in	O
the	O
SAM	O
-	O
bound	O
state	O
(	O
red	O
)	O
and	O
SsTsr3	O
(	O
blue	O
)	O
in	O
the	O
apo	O
state	O
.	O
	O
In	O
comparison	O
to	O
Tsr3	O
the	O
central	O
β	O
-	O
sheet	O
element	O
of	O
Trm10	O
is	O
extended	O
by	O
one	O
additional	O
β	O
-	O
strand	O
pairing	O
to	O
β2	O
.	O
	O
Furthermore	O
,	O
the	O
trefoil	O
knot	O
of	O
Trm10	O
is	O
not	O
as	O
deep	O
as	O
that	O
of	O
Tsr3	O
(	O
Figure	O
4D	O
).	O
	O
W73	O
is	O
highly	O
conserved	O
in	O
all	O
known	O
Tsr3	O
proteins	O
,	O
whereas	O
A76	O
can	O
be	O
replaced	O
by	O
other	O
hydrophobic	O
amino	O
acids	O
.	O
	O
(	O
A	O
)	O
Close	O
-	O
up	O
view	O
of	O
the	O
SAM	O
-	O
binding	O
pocket	O
of	O
VdTsr3	O
.	O
	O
Bound	O
SAM	O
was	O
modelled	O
based	O
on	O
the	O
X	O
-	O
ray	O
structure	O
of	O
the	O
Trm10	O
/	O
SAH	O
-	O
complex	O
(	O
pdb4jwf	O
).	O
	O
A	O
red	O
arrow	O
indicates	O
the	O
SAM	O
methyl	O
group	O
.	O
(	O
D	O
)	O
Binding	O
of	O
SAM	O
analogs	O
to	O
SsTsr3	O
.	O
	O
SsTsr3	O
bound	O
SAM	O
with	O
a	O
KD	O
of	O
6	O
.	O
5	O
μM	O
,	O
which	O
is	O
similar	O
to	O
SAM	O
-	O
KD	O
'	O
s	O
reported	O
for	O
several	O
SPOUT	O
-	O
class	O
methyltransferases	O
.	O
	O
5	O
′-	O
methylthioadenosin	O
—	O
the	O
reaction	O
product	O
after	O
the	O
acp	O
-	O
transfer	O
—	O
binds	O
only	O
∼	O
2	O
.	O
5	O
-	O
fold	O
weaker	O
(	O
KD	O
=	O
16	O
.	O
7	O
μM	O
)	O
compared	O
to	O
SAM	O
.	O
	O
Mutations	O
of	O
the	O
corresponding	O
residue	O
in	O
SsTsr3	O
to	O
A	O
(	O
D63	O
)	O
does	O
not	O
significantly	O
alter	O
the	O
SAM	O
-	O
binding	O
affinity	O
of	O
the	O
protein	O
(	O
KD	O
=	O
11	O
.	O
0	O
μM	O
).	O
	O
Analysis	O
of	O
the	O
electrostatic	O
surface	O
properties	O
of	O
VdTsr3	O
clearly	O
identified	O
positively	O
charged	O
surface	O
patches	O
in	O
the	O
vicinity	O
of	O
the	O
SAM	O
-	O
binding	O
site	O
suggesting	O
a	O
putative	O
RNA	O
-	O
binding	O
site	O
(	O
Figure	O
6A	O
).	O
	O
Its	O
negatively	O
charged	O
sulfate	O
group	O
might	O
mimic	O
an	O
RNA	O
backbone	O
phosphate	O
.	O
	O
In	O
order	O
to	O
explore	O
the	O
RNA	O
-	O
ligand	O
specificity	O
of	O
Tsr3	O
we	O
titrated	O
SsTsr3	O
prepared	O
in	O
RNase	O
-	O
free	O
form	O
with	O
5	O
′-	O
fluoresceine	O
-	O
labeled	O
RNA	O
and	O
determined	O
the	O
affinity	O
by	O
fluorescence	O
anisotropy	O
measurements	O
.	O
	O
A	O
single	O
stranded	O
oligoU	O
-	O
RNA	O
bound	O
with	O
a	O
10	O
-	O
fold	O
-	O
reduced	O
affinity	O
(	O
6	O
.	O
0	O
μM	O
).	O
	O
This	O
makes	O
it	O
unique	O
in	O
eukaryotic	O
rRNA	O
modification	O
.	O
	O
A	O
similar	O
modification	O
(	O
acp3U	O
)	O
was	O
identified	O
in	O
Haloferax	O
volcanii	O
and	O
corresponding	O
modified	O
nucleotides	O
were	O
also	O
shown	O
to	O
occur	O
in	O
other	O
archaea	O
.	O
	O
This	O
demonstrates	O
that	O
,	O
unlike	O
the	O
other	O
small	O
subunit	O
rRNA	O
base	O
modifications	O
,	O
the	O
acp	O
modification	O
is	O
required	O
for	O
efficient	O
pre	O
-	O
rRNA	O
processing	O
.	O
	O
After	O
structural	O
changes	O
,	O
possibly	O
driven	O
by	O
GTP	O
hydrolysis	O
,	O
which	O
go	O
together	O
with	O
the	O
formation	O
of	O
the	O
decoding	O
site	O
,	O
the	O
20S	O
pre	O
-	O
rRNA	O
becomes	O
accessible	O
for	O
Nob1	O
cleavage	O
at	O
site	O
D	O
.	O
This	O
also	O
involves	O
joining	O
of	O
pre	O
-	O
40S	O
and	O
60S	O
subunits	O
to	O
80S	O
-	O
like	O
particles	O
in	O
a	O
translation	O
-	O
like	O
cycle	O
promoted	O
by	O
eIF5B	O
.	O
	O
Thus	O
,	O
the	O
acp	O
transfer	O
to	O
m1Ψ1191	O
occurs	O
during	O
the	O
step	O
at	O
which	O
Rio2	O
leaves	O
the	O
pre	O
-	O
40S	O
particle	O
.	O
	O
The	O
current	O
data	O
together	O
with	O
the	O
finding	O
that	O
acp	O
modification	O
takes	O
place	O
at	O
the	O
very	O
last	O
step	O
in	O
pre	O
-	O
40S	O
subunit	O
maturation	O
indicate	O
that	O
the	O
acp	O
modification	O
probably	O
supports	O
the	O
formation	O
of	O
the	O
decoding	O
site	O
and	O
efficient	O
20S	O
pre	O
-	O
rRNA	O
D	O
-	O
site	O
cleavage	O
.	O
	O
Furthermore	O
,	O
our	O
structural	O
data	O
unravelled	O
how	O
the	O
regioselectivity	O
of	O
SAM	O
-	O
dependent	O
group	O
transfer	O
reactions	O
can	O
be	O
tuned	O
by	O
distinct	O
small	O
evolutionary	O
adaptions	O
of	O
the	O
ligand	O
binding	O
pocket	O
of	O
SAM	O
-	O
binding	O
enzymes	O
.	O
	O
In	O
addition	O
,	O
our	O
crystallographic	O
analyses	O
revealed	O
that	O
YfiR	O
binds	O
Vitamin	O
B6	O
(	O
VB6	O
)	O
or	O
L	O
-	O
Trp	O
at	O
a	O
YfiB	O
-	O
binding	O
site	O
and	O
that	O
both	O
VB6	O
and	O
L	O
-	O
Trp	O
are	O
able	O
to	O
reduce	O
YfiBL43P	B-mutant
-	O
induced	O
biofilm	O
formation	O
.	O
	O
An	O
increase	O
in	O
c	O
-	O
di	O
-	O
GMP	O
promotes	O
biofilm	O
formation	O
,	O
and	O
a	O
decrease	O
results	O
in	O
biofilm	O
degradation	O
(	O
Boehm	O
et	O
al	O
.,;	O
Duerig	O
et	O
al	O
.,;	O
Hickman	O
et	O
al	O
.,;	O
Jenal	O
,;	O
Romling	O
et	O
al	O
.,).	O
	O
The	O
c	O
-	O
di	O
-	O
GMP	O
level	O
is	O
regulated	O
by	O
two	O
reciprocal	O
enzyme	O
systems	O
,	O
namely	O
,	O
diguanylate	O
cyclases	O
(	O
DGCs	O
)	O
that	O
synthesize	O
c	O
-	O
di	O
-	O
GMP	O
and	O
phosphodiesterases	O
(	O
PDEs	O
)	O
that	O
hydrolyze	O
c	O
-	O
di	O
-	O
GMP	O
(	O
Kulasakara	O
et	O
al	O
.,;	O
Ross	O
et	O
al	O
.,;	O
Ross	O
et	O
al	O
.,).	O
Many	O
of	O
these	O
enzymes	O
are	O
multiple	O
-	O
domain	O
proteins	O
containing	O
a	O
variable	O
N	O
-	O
terminal	O
domain	O
that	O
commonly	O
acts	O
as	O
a	O
signal	O
sensor	O
or	O
transduction	O
module	O
,	O
followed	O
by	O
the	O
relatively	O
conserved	O
GGDEF	O
motif	O
in	O
DGCs	O
or	O
EAL	O
/	O
HD	O
-	O
GYP	O
domains	O
in	O
PDEs	O
(	O
Hengge	O
,;	O
Navarro	O
et	O
al	O
.,;	O
Schirmer	O
and	O
Jenal	O
,).	O
	O
YfiN	O
is	O
an	O
integral	O
inner	O
-	O
membrane	O
protein	O
with	O
two	O
potential	O
transmembrane	O
helices	O
,	O
a	O
periplasmic	O
Per	O
-	O
Arnt	O
-	O
Sim	O
(	O
PAS	O
)	O
domain	O
,	O
and	O
cytosolic	O
domains	O
containing	O
a	O
HAMP	O
domain	O
(	O
mediate	O
input	O
-	O
output	O
signaling	O
in	O
histidine	O
kinases	O
,	O
adenylyl	O
cyclases	O
,	O
methyl	O
-	O
accepting	O
chemotaxis	O
proteins	O
,	O
and	O
phosphatases	O
)	O
and	O
a	O
C	O
-	O
terminal	O
GGDEF	O
domain	O
indicating	O
a	O
DGC	O
’	O
s	O
function	O
(	O
Giardina	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,).	O
	O
YfiN	O
is	O
repressed	O
by	O
specific	O
interaction	O
between	O
its	O
periplasmic	O
PAS	O
domain	O
and	O
the	O
periplasmic	O
protein	O
YfiR	O
(	O
Malone	O
et	O
al	O
.,).	O
	O
After	O
the	O
sequestration	O
of	O
YfiR	O
by	O
YfiB	O
,	O
the	O
c	O
-	O
di	O
-	O
GMP	O
produced	O
by	O
activated	O
YfiN	O
increases	O
the	O
biosynthesis	O
of	O
the	O
Pel	O
and	O
Psl	O
EPSs	O
,	O
resulting	O
in	O
the	O
appearance	O
of	O
the	O
SCV	O
phenotype	O
,	O
which	O
indicates	O
enhanced	O
biofilm	O
formation	O
(	O
Malone	O
et	O
al	O
.,).	O
	O
Recently	O
,	O
we	O
solved	O
the	O
crystal	O
structure	O
of	O
YfiR	O
in	O
both	O
the	O
non	O
-	O
oxidized	O
and	O
the	O
oxidized	O
states	O
,	O
revealing	O
breakage	O
/	O
formation	O
of	O
one	O
disulfide	O
bond	O
(	O
Cys71	O
-	O
Cys110	O
)	O
and	O
local	O
conformational	O
change	O
around	O
the	O
other	O
one	O
(	O
Cys145	O
-	O
Cys152	O
),	O
indicating	O
that	O
Cys145	O
-	O
Cys152	O
plays	O
an	O
important	O
role	O
in	O
maintaining	O
the	O
correct	O
folding	O
of	O
YfiR	O
(	O
Yang	O
et	O
al	O
.,).	O
	O
The	O
“	O
back	O
to	O
back	O
”	O
dimer	O
.	O
	O
The	O
dimerization	O
occurs	O
mainly	O
via	O
hydrophobic	O
interactions	O
formed	O
by	O
A37	O
and	O
I40	O
on	O
the	O
α1	O
helices	O
,	O
L50	O
on	O
the	O
β1	O
strands	O
,	O
and	O
W55	O
on	O
the	O
β2	O
strands	O
of	O
both	O
molecules	O
,	O
making	O
a	O
hydrophobic	O
interacting	O
core	O
(	O
Fig	O
.	O
2A	O
–	O
C	O
).	O
	O
The	O
“	O
back	O
to	O
back	O
”	O
dimer	O
presents	O
a	O
Y	O
shape	O
.	O
	O
Overall	O
structure	O
of	O
the	O
YfiB	O
-	O
YfiR	O
complex	O
and	O
the	O
conserved	O
surface	O
in	O
YfiR	O
.	O
(	O
A	O
)	O
The	O
overall	O
structure	O
of	O
the	O
YfiB	O
-	O
YfiR	O
complex	O
.	O
	O
Two	O
interacting	O
regions	O
are	O
highlighted	O
by	O
red	O
rectangles	O
.	O
(	O
B	O
)	O
Structural	O
superposition	O
of	O
apo	O
YfiB	O
and	O
YfiR	O
-	O
bound	O
YfiBL43P	B-mutant
.	O
	O
The	O
YfiB	O
-	O
YfiR	O
complex	O
is	O
a	O
2	O
:	O
2	O
heterotetramer	O
(	O
Fig	O
.	O
3A	O
)	O
in	O
which	O
the	O
YfiR	O
dimer	O
is	O
clamped	O
by	O
two	O
separated	O
YfiBL43P	B-mutant
molecules	O
with	O
a	O
total	O
buried	O
surface	O
area	O
of	O
3161	O
.	O
2	O
Å2	O
.	O
	O
Additionally	O
,	O
three	O
hydrophobic	O
anchoring	O
sites	O
exist	O
in	O
region	O
I	O
.	O
The	O
residues	O
F48	O
and	O
W55	O
of	O
YfiB	O
are	O
inserted	O
into	O
the	O
hydrophobic	O
cores	O
mainly	O
formed	O
by	O
the	O
main	O
chain	O
and	O
side	O
chain	O
carbon	O
atoms	O
of	O
residues	O
S57	O
/	O
Q88	O
/	O
A89	O
/	O
N90	O
and	O
R60	O
/	O
R175	O
/	O
H177	O
of	O
YfiR	O
,	O
respectively	O
;	O
and	O
F57	O
of	O
YfiB	O
is	O
inserted	O
into	O
the	O
hydrophobic	O
pocket	O
formed	O
by	O
L166	O
/	O
I169	O
/	O
V176	O
/	O
P178	O
/	O
L181	O
of	O
YfiR	O
(	O
Fig	O
.	O
3D	O
-	O
I	O
(	O
ii	O
)).	O
	O
This	O
suggests	O
that	O
the	O
N	O
-	O
terminus	O
of	O
YfiB	O
plays	O
an	O
important	O
role	O
in	O
forming	O
the	O
dimeric	O
YfiB	O
in	O
solution	O
and	O
that	O
the	O
conformational	O
change	O
of	O
residue	O
L43	O
is	O
associated	O
with	O
the	O
stretch	O
of	O
the	O
N	O
-	O
terminus	O
and	O
opening	O
of	O
the	O
dimer	O
.	O
	O
The	O
PG	O
-	O
binding	O
site	O
of	O
YfiB	O
	O
In	O
the	O
YfiB	O
-	O
YfiR	O
complex	O
,	O
one	O
sulfate	O
ion	O
is	O
found	O
at	O
the	O
bottom	O
of	O
each	O
YfiBL43P	B-mutant
molecule	O
(	O
Fig	O
.	O
3A	O
)	O
and	O
forms	O
a	O
strong	O
hydrogen	O
bond	O
with	O
D102	O
of	O
YfiBL43P	B-mutant
(	O
Fig	O
.	O
4A	O
and	O
4C	O
).	O
	O
Moreover	O
,	O
a	O
water	O
molecule	O
was	O
found	O
to	O
bridge	O
the	O
sulfate	O
ion	O
and	O
the	O
side	O
chains	O
of	O
N67	O
and	O
D102	O
,	O
strengthening	O
the	O
hydrogen	O
bond	O
network	O
(	O
Fig	O
.	O
4C	O
).	O
	O
The	O
results	O
indicated	O
that	O
the	O
PG	O
-	O
binding	O
affinity	O
of	O
YfiBL43P	B-mutant
is	O
65	O
.	O
5	O
μmol	O
/	O
L	O
,	O
which	O
is	O
about	O
16	O
-	O
fold	O
stronger	O
than	O
that	O
of	O
wild	O
-	O
type	O
YfiB	O
(	O
Kd	O
=	O
1	O
.	O
1	O
mmol	O
/	O
L	O
)	O
(	O
Fig	O
.	O
4E	O
–	O
F	O
).	O
	O
The	O
relative	O
optical	O
density	O
is	O
represented	O
as	O
curves	O
.	O
	O
Wild	O
-	O
type	O
YfiB	O
is	O
used	O
as	O
negative	O
control	O
.	O
	O
Previous	O
studies	O
suggested	O
that	O
in	O
response	O
to	O
cell	O
stress	O
,	O
YfiB	O
in	O
the	O
outer	O
membrane	O
sequesters	O
the	O
periplasmic	O
protein	O
YfiR	O
,	O
releasing	O
its	O
inhibition	O
of	O
YfiN	O
on	O
the	O
inner	O
membrane	O
and	O
thus	O
inducing	O
the	O
diguanylate	O
cyclase	O
activity	O
of	O
YfiN	O
to	O
allow	O
c	O
-	O
di	O
-	O
GMP	O
production	O
(	O
Giardina	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,).	O
	O
Here	O
,	O
we	O
report	O
the	O
crystal	O
structures	O
of	O
YfiB	O
alone	O
and	O
an	O
active	O
mutant	O
YfiBL43P	B-mutant
in	O
complex	O
with	O
YfiR	O
,	O
indicating	O
that	O
YfiR	O
forms	O
a	O
2	O
:	O
2	O
complex	O
with	O
YfiB	O
via	O
a	O
region	O
composed	O
of	O
conserved	O
residues	O
.	O
	O
Our	O
structural	O
data	O
analysis	O
shows	O
that	O
the	O
activated	O
YfiB	O
has	O
an	O
N	O
-	O
terminal	O
portion	O
that	O
is	O
largely	O
altered	O
,	O
adopting	O
a	O
stretched	O
conformation	O
compared	O
with	O
the	O
compact	O
conformation	O
of	O
the	O
apo	O
YfiB	O
.	O
The	O
apo	O
YfiB	O
structure	O
constructed	O
beginning	O
at	O
residue	O
34	O
has	O
a	O
compact	O
conformation	O
of	O
approximately	O
45	O
Å	O
in	O
length	O
.	O
	O
In	O
this	O
model	O
,	O
in	O
response	O
to	O
a	O
particular	O
cell	O
stress	O
that	O
is	O
yet	O
to	O
be	O
identified	O
,	O
the	O
dimeric	O
YfiB	O
is	O
activated	O
from	O
a	O
compact	O
,	O
inactive	O
conformation	O
to	O
a	O
stretched	O
conformation	O
,	O
which	O
possesses	O
increased	O
PG	O
binding	O
affinity	O
.	O
	O
Homologs	O
of	O
the	O
YfiBNR	O
system	O
are	O
functionally	O
conserved	O
in	O
P	O
.	O
aeruginosa	O
(	O
Malone	O
et	O
al	O
.,;	O
Malone	O
et	O
al	O
.,),	O
E	O
.	O
coli	O
(	O
Hufnagel	O
et	O
al	O
.,;	O
Raterman	O
et	O
al	O
.,;	O
Sanchez	O
-	O
Torres	O
et	O
al	O
.,),	O
K	O
.	O
pneumonia	O
(	O
Huertas	O
et	O
al	O
.,)	O
and	O
Y	O
.	O
pestis	O
(	O
Ren	O
et	O
al	O
.,),	O
where	O
they	O
affect	O
c	O
-	O
di	O
-	O
GMP	O
production	O
and	O
biofilm	O
formation	O
.	O
	O
High	O
-	O
resolution	O
structures	O
of	O
oligomers	O
formed	O
by	O
the	O
β	O
-	O
amyloid	O
peptide	O
Aβ	O
are	O
needed	O
to	O
understand	O
the	O
molecular	O
basis	O
of	O
Alzheimer	O
’	O
s	O
disease	O
and	O
develop	O
therapies	O
.	O
	O
Over	O
the	O
last	O
two	O
decades	O
the	O
role	O
of	O
Aβ	O
oligomers	O
in	O
the	O
pathophysiology	O
of	O
Alzheimer	O
’	O
s	O
disease	O
has	O
begun	O
to	O
unfold	O
.	O
	O
Aβ	O
isolated	O
from	O
the	O
brains	O
of	O
young	O
plaque	O
-	O
free	O
Tg2576	O
mice	O
forms	O
a	O
mixture	O
of	O
low	O
molecular	O
weight	O
oligomers	O
.	O
	O
Smaller	O
oligomers	O
with	O
molecular	O
weights	O
consistent	O
with	O
trimers	O
,	O
hexamers	O
,	O
and	O
nonamers	O
were	O
also	O
identified	O
within	O
the	O
mixture	O
of	O
low	O
molecular	O
weight	O
oligomers	O
.	O
	O
A	O
type	O
of	O
large	O
oligomers	O
called	O
annular	O
protofibrils	O
(	O
APFs	O
)	O
have	O
also	O
been	O
observed	O
in	O
the	O
brains	O
of	O
transgenic	O
mice	O
and	O
isolated	O
from	O
the	O
brains	O
of	O
Alzheimer	O
’	O
s	O
patients	O
.	O
	O
Lashuel	O
et	O
al	O
.	O
observed	O
APFs	O
with	O
an	O
outer	O
diameter	O
that	O
ranged	O
from	O
7	O
–	O
10	O
nm	O
and	O
an	O
inner	O
diameter	O
that	O
ranged	O
from	O
1	O
.	O
5	O
–	O
2	O
nm	O
,	O
consistent	O
with	O
molecular	O
weights	O
of	O
150	O
–	O
250	O
kDa	O
.	O
	O
Kayed	O
et	O
al	O
.	O
observed	O
APFs	O
with	O
an	O
outer	O
diameter	O
that	O
ranged	O
from	O
8	O
–	O
25	O
nm	O
,	O
which	O
were	O
composed	O
of	O
small	O
spherical	O
Aβ	O
oligomers	O
,	O
3	O
–	O
5	O
nm	O
in	O
diameter	O
.	O
	O
Sequestering	O
Aβ	O
within	O
the	O
affibody	O
prevents	O
its	O
fibrilization	O
and	O
reduces	O
its	O
neurotoxicity	O
,	O
providing	O
evidence	O
that	O
the	O
β	O
-	O
hairpin	O
structure	O
may	O
contribute	O
to	O
the	O
ability	O
of	O
Aβ	O
to	O
form	O
neurotoxic	O
oligomers	O
.	O
	O
(	O
A	O
)	O
Cartoon	O
illustrating	O
the	O
design	O
of	O
peptides	O
1	O
and	O
2	O
and	O
their	O
relationship	O
to	O
an	O
Aβ17	O
–	O
36	O
β	O
-	O
hairpin	O
.	O
	O
Peptide	B-mutant
2	I-mutant
contains	O
a	O
methionine	O
residue	O
at	O
position	O
35	O
and	O
an	O
Aβ24	O
–	O
29	O
loop	O
with	O
residues	O
24	O
and	O
29	O
(	O
Val	O
and	O
Gly	O
)	O
mutated	O
to	O
cysteine	O
and	O
linked	O
by	O
a	O
disulfide	O
bond	O
(	O
Figure	O
1C	O
).	O
	O
To	O
address	O
this	O
issue	O
,	O
we	O
next	O
incorporated	O
a	O
disulfide	O
bond	O
between	O
residues	O
24	O
and	O
29	O
as	O
a	O
conformational	O
constraint	O
that	O
serves	O
as	O
a	O
surrogate	O
for	O
δOrn	O
.	O
	O
We	O
mutated	O
these	O
residues	O
because	O
they	O
occupy	O
the	O
same	O
position	O
as	O
the	O
δOrn	O
that	O
connects	O
D23	O
and	O
A30	O
in	O
peptide	B-mutant
1	I-mutant
.	O
	O
In	O
synthesizing	O
peptides	B-mutant
2	I-mutant
and	I-mutant
4	I-mutant
we	O
formed	O
the	O
disulfide	O
linkage	O
after	O
macrolactamization	O
and	O
deprotection	O
of	O
the	O
acid	O
-	O
labile	O
side	O
chain	O
protecting	O
groups	O
.	O
	O
Crystal	O
diffraction	O
data	O
for	O
peptides	B-mutant
4	I-mutant
and	I-mutant
2	I-mutant
were	O
collected	O
in	O
-	O
house	O
with	O
a	O
Rigaku	O
MicroMax	O
007HF	O
X	O
-	O
ray	O
diffractometer	O
at	O
1	O
.	O
54	O
Å	O
wavelength	O
.	O
	O
Data	O
for	O
peptides	B-mutant
4	I-mutant
and	I-mutant
2	I-mutant
were	O
scaled	O
and	O
merged	O
using	O
XDS	O
.	O
	O
X	O
-	O
ray	O
Crystallographic	O
Structure	O
of	O
Peptide	B-mutant
2	I-mutant
and	O
the	O
Oligomers	O
It	O
Forms	O
	O
The	O
B	O
values	O
for	O
the	O
loops	O
are	O
large	O
,	O
indicating	O
that	O
the	O
loops	O
are	O
dynamic	O
and	O
not	O
well	O
ordered	O
.	O
	O
Thus	O
,	O
the	O
differences	O
in	O
backbone	O
geometry	O
and	O
side	O
chain	O
rotamers	O
among	O
the	O
loops	O
are	O
likely	O
of	O
little	O
significance	O
and	O
should	O
be	O
interpreted	O
with	O
caution	O
.	O
	O
Like	O
peptide	B-mutant
1	I-mutant
,	O
peptide	B-mutant
2	I-mutant
forms	O
a	O
triangular	O
trimer	O
,	O
and	O
four	O
trimers	O
assemble	O
to	O
form	O
a	O
dodecamer	O
.	O
	O
In	O
the	O
higher	O
-	O
order	O
assembly	O
of	O
the	O
dodecamers	O
formed	O
by	O
peptide	B-mutant
2	I-mutant
a	O
new	O
structure	O
emerges	O
,	O
not	O
seen	O
in	O
peptide	B-mutant
1	I-mutant
,	O
an	O
annular	O
pore	O
consisting	O
of	O
five	O
dodecamers	O
.	O
	O
The	O
trimer	O
maintains	O
all	O
of	O
the	O
same	O
stabilizing	O
contacts	O
as	O
those	O
of	O
peptide	B-mutant
1	I-mutant
.	O
	O
In	O
the	O
crystal	O
lattice	O
,	O
each	O
F20	O
face	O
of	O
one	O
dodecamer	O
packs	O
against	O
an	O
F20	O
face	O
of	O
another	O
dodecamer	O
.	O
	O
Jeffamine	O
M	O
-	O
600	O
is	O
a	O
polypropylene	O
glycol	O
derivative	O
with	O
a	O
2	O
-	O
methoxyethoxy	O
unit	O
at	O
one	O
end	O
and	O
a	O
2	O
-	O
aminopropyl	O
unit	O
at	O
the	O
other	O
end	O
.	O
	O
Hydrophobic	O
packing	O
between	O
the	O
F20	O
faces	O
of	O
trimers	O
displayed	O
on	O
the	O
outer	O
surface	O
of	O
each	O
dodecamer	O
stabilizes	O
the	O
porelike	O
assembly	O
.	O
	O
The	O
eclipsed	O
interfaces	O
occur	O
between	O
dodecamers	O
1	O
and	O
2	O
,	O
1	O
and	O
5	O
,	O
and	O
3	O
and	O
4	O
,	O
as	O
shown	O
in	O
Figure	O
5A	O
.	O
	O
The	O
crystallographic	O
assembly	O
of	O
peptide	B-mutant
2	I-mutant
into	O
a	O
trimer	O
,	O
dodecamer	O
,	O
and	O
annular	O
pore	O
provides	O
a	O
model	O
for	O
the	O
assembly	O
of	O
the	O
full	O
-	O
length	O
Aβ	O
peptide	O
to	O
form	O
oligomers	O
.	O
	O
In	O
this	O
model	O
Aβ	O
folds	O
to	O
form	O
a	O
β	O
-	O
hairpin	O
comprising	O
the	O
hydrophobic	O
central	O
and	O
C	O
-	O
terminal	O
regions	O
.	O
	O
Three	O
β	O
-	O
hairpins	O
assemble	O
to	O
form	O
a	O
trimer	O
,	O
and	O
four	O
trimers	O
assemble	O
to	O
form	O
a	O
dodecamer	O
.	O
	O
The	O
model	O
put	O
forth	O
in	O
Figure	O
6	O
is	O
consistent	O
with	O
the	O
current	O
understanding	O
of	O
endogenous	O
Aβ	O
oligomerization	O
and	O
explains	O
at	O
atomic	O
resolution	O
many	O
key	O
observations	O
about	O
Aβ	O
oligomers	O
.	O
	O
Fibrillar	O
and	O
nonfibrillar	O
oligomers	O
have	O
structurally	O
distinct	O
characteristics	O
,	O
which	O
are	O
reflected	O
in	O
their	O
reactivity	O
with	O
the	O
fibril	O
-	O
specific	O
OC	O
antibody	O
and	O
the	O
oligomer	O
-	O
specific	O
A11	O
antibody	O
.	O
	O
At	O
this	O
point	O
,	O
we	O
can	O
only	O
speculate	O
whether	O
the	O
trimer	O
and	O
dodecamer	O
formed	O
by	O
peptide	B-mutant
2	I-mutant
share	O
structural	O
similarities	O
to	O
Aβ	O
trimers	O
and	O
Aβ	O
*	O
56	O
,	O
as	O
little	O
is	O
known	O
about	O
the	O
structure	O
of	O
Aβ	O
trimers	O
and	O
Aβ	O
*	O
56	O
.	O
	O
These	O
two	O
modes	O
of	O
assembly	O
might	O
reflect	O
a	O
dynamic	O
interaction	O
between	O
dodecamers	O
,	O
which	O
could	O
permit	O
assemblies	O
of	O
more	O
dodecamers	O
into	O
larger	O
annular	O
pores	O
.	O
	O
Preliminary	O
attempts	O
to	O
study	O
these	O
species	O
by	O
SEC	O
and	O
SDS	O
-	O
PAGE	O
have	O
not	O
provided	O
a	O
clear	O
measure	O
of	O
the	O
structures	O
formed	O
in	O
solution	O
.	O
	O
Our	O
approach	O
of	O
constraining	O
Aβ17	O
–	O
36	O
into	O
a	O
β	O
-	O
hairpin	O
conformation	O
and	O
blocking	O
aggregation	O
with	O
an	O
N	O
-	O
methyl	O
group	O
has	O
allowed	O
us	O
to	O
crystallize	O
a	O
large	O
fragment	O
of	O
what	O
is	O
generally	O
considered	O
to	O
be	O
an	O
uncrystallizable	O
peptide	O
.	O
	O
Ligands	O
that	O
regulate	O
the	O
dynamics	O
and	O
stability	O
of	O
the	O
coactivator	O
‐	O
binding	O
site	O
in	O
the	O
C	O
‐	O
terminal	O
ligand	O
‐	O
binding	O
domain	O
,	O
called	O
activation	O
function	O
‐	O
2	O
(	O
AF	O
‐	O
2	O
),	O
showed	O
similar	O
activity	O
profiles	O
in	O
different	O
cell	O
types	O
.	O
	O
Such	O
ligands	O
induced	O
breast	O
cancer	O
cell	O
proliferation	O
in	O
a	O
manner	O
that	O
was	O
predicted	O
by	O
the	O
canonical	O
recruitment	O
of	O
the	O
coactivators	O
NCOA1	O
/	O
2	O
/	O
3	O
and	O
induction	O
of	O
the	O
GREB1	O
proliferative	O
gene	O
.	O
	O
For	O
example	O
,	O
selective	O
estrogen	O
receptor	O
modulators	O
(	O
SERMs	O
)	O
such	O
as	O
tamoxifen	O
(	O
Nolvadex	O
®;	O
AstraZeneca	O
)	O
or	O
raloxifene	O
(	O
Evista	O
®;	O
Eli	O
Lilly	O
)	O
(	O
Fig	O
1A	O
)	O
block	O
the	O
ERα	O
‐	O
mediated	O
proliferative	O
effects	O
of	O
the	O
native	O
estrogen	O
,	O
17β	O
‐	O
estradiol	O
(	O
E2	O
),	O
on	O
breast	O
cancer	O
cells	O
,	O
but	O
promote	O
beneficial	O
estrogenic	O
effects	O
on	O
bone	O
mineral	O
density	O
and	O
adverse	O
estrogenic	O
effects	O
such	O
as	O
uterine	O
proliferation	O
,	O
fatty	O
liver	O
,	O
or	O
stroke	O
(	O
Frolik	O
et	O
al	O
,	O
1996	O
;	O
Fisher	O
et	O
al	O
,	O
1998	O
;	O
McDonnell	O
et	O
al	O
,	O
2002	O
;	O
Jordan	O
,	O
2003	O
).	O
	O
E2	O
‐	O
rings	O
are	O
numbered	O
A	O
‐	O
D	O
.	O
The	O
E	O
‐	O
ring	O
is	O
the	O
common	O
site	O
of	O
attachment	O
for	O
BSC	O
found	O
in	O
many	O
SERMS	O
.	O
	O
Linear	O
causality	O
model	O
for	O
ERα	O
‐	O
mediated	O
cell	O
proliferation	O
.	O
	O
AF	O
‐	O
1	O
binds	O
a	O
separate	O
surface	O
on	O
these	O
coactivators	O
(	O
Webb	O
et	O
al	O
,	O
1998	O
;	O
Yi	O
et	O
al	O
,	O
2015	O
).	O
	O
However	O
,	O
ERα	O
‐	O
mediated	O
proliferative	O
responses	O
vary	O
in	O
a	O
ligand	O
‐	O
dependent	O
manner	O
(	O
Srinivasan	O
et	O
al	O
,	O
2013	O
);	O
thus	O
,	O
it	O
is	O
not	O
known	O
whether	O
this	O
canonical	O
model	O
is	O
widely	O
applicable	O
across	O
diverse	O
ERα	O
ligands	O
.	O
	O
In	O
this	O
signaling	O
model	O
,	O
multiple	O
coregulator	O
binding	O
events	O
and	O
target	O
genes	O
(	O
Won	O
Jeong	O
et	O
al	O
,	O
2012	O
;	O
Nwachukwu	O
et	O
al	O
,	O
2014	O
),	O
LBD	O
conformation	O
,	O
nucleocytoplasmic	O
shuttling	O
,	O
the	O
occupancy	O
and	O
dynamics	O
of	O
DNA	O
binding	O
,	O
and	O
other	O
biophysical	O
features	O
could	O
contribute	O
independently	O
to	O
cell	O
proliferation	O
(	O
Lickwar	O
et	O
al	O
,	O
2012	O
).	O
	O
To	O
test	O
these	O
signaling	O
models	O
,	O
we	O
profiled	O
a	O
diverse	O
library	O
of	O
ERα	O
ligands	O
using	O
systems	O
biology	O
approaches	O
to	O
X	O
‐	O
ray	O
crystallography	O
and	O
chemical	O
biology	O
(	O
Srinivasan	O
et	O
al	O
,	O
2013	O
),	O
including	O
a	O
series	O
of	O
quantitative	O
bioassays	O
for	O
ERα	O
function	O
that	O
were	O
statistically	O
robust	O
and	O
reproducible	O
,	O
based	O
on	O
the	O
Z	O
’‐	O
statistic	O
(	O
Fig	O
EV1A	O
and	O
B	O
;	O
see	O
Materials	O
and	O
Methods	O
).	O
	O
Structure	O
of	O
the	O
E2	O
‐	O
bound	O
ERα	O
LBD	O
in	O
complex	O
with	O
an	O
NCOA2	O
peptide	O
of	O
(	O
PDB	O
1GWR	O
).	O
	O
In	O
cluster	O
1	O
,	O
the	O
first	O
three	O
comparisons	O
(	O
rows	O
)	O
showed	O
significant	O
positive	O
correlations	O
(	O
F	O
‐	O
test	O
for	O
nonzero	O
slope	O
,	O
P	O
≤	O
0	O
.	O
05	O
).	O
	O
−,	O
significant	O
correlations	O
lost	O
upon	O
deletion	O
of	O
AB	O
or	O
F	O
domains	O
.	O
	O
Tamoxifen	O
depends	O
on	O
AF	O
‐	O
1	O
for	O
its	O
cell	O
‐	O
specific	O
activity	O
(	O
Sakamoto	O
et	O
al	O
,	O
2002	O
);	O
therefore	O
,	O
we	O
asked	O
whether	O
cell	O
‐	O
specific	O
signaling	O
observed	O
here	O
is	O
due	O
to	O
a	O
similar	O
dependence	O
on	O
AF	O
‐	O
1	O
for	O
activity	O
(	O
Fig	O
EV1	O
).	O
	O
Thus	O
,	O
the	O
strength	O
of	O
AF	O
‐	O
1	O
signaling	O
does	O
not	O
determine	O
cell	O
‐	O
specific	O
signaling	O
.	O
	O
Identifying	O
cell	O
‐	O
specific	O
signaling	O
clusters	O
in	O
ERα	O
ligand	O
classes	O
	O
The	O
side	O
chain	O
of	O
OBHS	O
‐	O
BSC	O
analogs	O
induces	O
cell	O
‐	O
specific	O
signaling	O
	O
In	O
panel	O
(	O
D	O
),	O
L	O
‐	O
Luc	O
ERα	O
‐	O
WT	O
activity	O
from	O
panel	O
(	O
B	O
)	O
is	O
shown	O
for	O
comparison	O
.	O
	O
Thus	O
,	O
examining	O
the	O
correlated	O
patterns	O
of	O
ERα	O
activity	O
within	O
each	O
scaffold	O
demonstrates	O
that	O
an	O
extended	O
side	O
chain	O
is	O
not	O
required	O
for	O
cell	O
‐	O
specific	O
signaling	O
.	O
	O
Deletion	O
of	O
the	O
AB	O
or	O
F	O
domain	O
altered	O
correlations	O
for	O
six	O
of	O
the	O
eight	O
scaffolds	O
in	O
this	O
cluster	O
(	O
2	O
,	O
5	O
‐	O
DTP	O
,	O
3	O
,	O
4	O
‐	O
DTP	O
,	O
S	O
‐	O
OBHS	O
‐	O
3	O
,	O
WAY	O
‐	O
D	O
,	O
WAY	O
dimer	O
,	O
and	O
cyclofenil	O
‐	O
ASC	O
)	O
(	O
Fig	O
3D	O
lanes	O
5	O
–	O
12	O
).	O
	O
Thus	O
,	O
in	O
cluster	O
2	O
,	O
AF	O
‐	O
1	O
substantially	O
modulated	O
the	O
specificity	O
of	O
ligands	O
with	O
cell	O
‐	O
specific	O
activity	O
(	O
Fig	O
3D	O
lanes	O
5	O
–	O
12	O
).	O
	O
To	O
determine	O
whether	O
ligand	O
classes	O
control	O
expression	O
of	O
native	O
ERα	O
target	O
genes	O
through	O
the	O
canonical	O
linear	O
signaling	O
pathway	O
,	O
we	O
performed	O
pairwise	O
linear	O
regression	O
analyses	O
using	O
ERα	O
–	O
NCOA1	O
/	O
2	O
/	O
3	O
interactions	O
in	O
M2H	O
assay	O
as	O
independent	O
predictors	O
of	O
GREB1	O
expression	O
(	O
the	O
dependent	O
variable	O
)	O
(	O
Figs	O
EV1	O
and	O
EV2A	O
,	O
F	O
–	O
H	O
).	O
	O
For	O
clusters	O
2	O
and	O
3	O
,	O
GREB1	O
activity	O
was	O
generally	O
not	O
predicted	O
by	O
NCOA1	O
/	O
2	O
/	O
3	O
recruitment	O
.	O
	O
However	O
,	O
ligand	O
‐	O
induced	O
GREB1	O
levels	O
were	O
generally	O
not	O
determined	O
by	O
NCOA1	O
/	O
2	O
/	O
3	O
recruitment	O
(	O
Fig	O
3E	O
lanes	O
5	O
–	O
19	O
),	O
consistent	O
with	O
an	O
alternate	O
causality	O
model	O
(	O
Fig	O
1E	O
).	O
	O
Out	O
of	O
11	O
indirect	O
modulator	O
series	O
in	O
cluster	O
2	O
or	O
3	O
,	O
only	O
the	O
S	O
‐	O
OBHS	O
‐	O
3	O
class	O
had	O
NCOA1	O
/	O
2	O
/	O
3	O
recruitment	O
profiles	O
that	O
predicted	O
GREB1	O
levels	O
(	O
Fig	O
3E	O
lane	O
12	O
).	O
	O
With	O
the	O
OBHS	O
‐	O
N	O
compounds	O
,	O
NCOA3	O
and	O
GREB1	O
showed	O
near	O
perfect	O
prediction	O
of	O
proliferation	O
(	O
Fig	O
EV3G	O
),	O
with	O
unexplained	O
variance	O
similar	O
to	O
the	O
noise	O
in	O
the	O
assays	O
.	O
	O
Out	O
of	O
15	O
ligand	O
series	O
in	O
these	O
clusters	O
,	O
only	O
2	O
,	O
5	O
‐	O
DTP	O
analogs	O
induced	O
a	O
proliferative	O
response	O
that	O
was	O
predicted	O
by	O
GREB1	O
levels	O
,	O
which	O
were	O
not	O
determined	O
by	O
NCOA1	O
/	O
2	O
/	O
3	O
recruitment	O
(	O
Fig	O
3E	O
and	O
F	O
lane	O
10	O
).	O
	O
Similarly	O
,	O
S	O
‐	O
OBHS	O
‐	O
3	O
,	O
cyclofenil	O
‐	O
ASC	O
,	O
and	O
OBHS	O
‐	O
ASC	O
had	O
positively	O
correlated	O
NCOA1	O
/	O
2	O
/	O
3	O
recruitment	O
and	O
GREB1	O
levels	O
,	O
but	O
none	O
of	O
these	O
activities	O
determined	O
their	O
proliferative	O
effects	O
(	O
Fig	O
3E	O
and	O
F	O
lanes	O
11	O
–	O
12	O
and	O
18	O
).	O
	O
NCOA3	O
occupancy	O
at	O
GREB1	O
is	O
statistically	O
robust	O
but	O
does	O
not	O
predict	O
transcriptional	O
activity	O
	O
All	O
direct	O
modulator	O
and	O
two	O
indirect	O
modulator	O
scaffolds	O
(	O
OBHS	O
and	O
S	O
‐	O
OBHS	O
‐	O
3	O
)	O
lacked	O
ERβ	O
agonist	O
activity	O
.	O
	O
ERα	O
activity	O
of	O
2	O
,	O
5	O
‐	O
DTP	O
and	O
cyclofenil	O
analogs	O
correlates	O
with	O
E	O
‐	O
Luc	O
activity	O
.	O
	O
Therefore	O
,	O
we	O
examined	O
another	O
50	O
LBD	O
structures	O
containing	O
ligands	O
in	O
clusters	O
2	O
and	O
3	O
.	O
	O
Ligands	O
in	O
cluster	O
2	O
and	O
cluster	O
3	O
showed	O
conformational	O
heterogeneity	O
in	O
parts	O
of	O
the	O
scaffold	O
that	O
were	O
directed	O
toward	O
multiple	O
regions	O
of	O
the	O
receptor	O
including	O
h3	O
,	O
h8	O
,	O
h11	O
,	O
h12	O
,	O
and	O
/	O
or	O
the	O
β	O
‐	O
sheets	O
(	O
Fig	O
EV5C	O
–	O
G	O
).	O
	O
Hierarchical	O
clustering	O
revealed	O
that	O
many	O
of	O
the	O
2	O
,	O
5	O
‐	O
DTP	O
analogs	O
recapitulated	O
most	O
of	O
the	O
peptide	O
recruitment	O
and	O
dismissal	O
patterns	O
observed	O
with	O
E2	O
(	O
Fig	O
6H	O
).	O
	O
Also	O
,	O
we	O
have	O
used	O
siRNA	O
screening	O
to	O
identify	O
a	O
number	O
of	O
coregulators	O
required	O
for	O
ERα	O
‐	O
mediated	O
repression	O
of	O
the	O
IL	O
‐	O
6	O
gene	O
(	O
Nwachukwu	O
et	O
al	O
,	O
2014	O
).	O
	O
Some	O
of	O
these	O
ligands	O
altered	O
the	O
shape	O
of	O
the	O
AF	O
‐	O
2	O
surface	O
by	O
perturbing	O
the	O
h3	O
–	O
h12	O
interface	O
,	O
thus	O
providing	O
a	O
route	O
to	O
new	O
SERM	O
‐	O
like	O
activity	O
profiles	O
by	O
combining	O
indirect	O
and	O
direct	O
modulation	O
of	O
receptor	O
structure	O
.	O
	O
Incorporation	O
of	O
statistical	O
approaches	O
to	O
understand	O
relationships	O
between	O
structure	O
and	O
signaling	O
variables	O
moves	O
us	O
toward	O
predictive	O
models	O
for	O
complex	O
ERα	O
‐	O
mediated	O
responses	O
such	O
as	O
in	O
vivo	O
uterine	O
proliferation	O
or	O
tumor	O
growth	O
,	O
and	O
more	O
generally	O
toward	O
structure	O
‐	O
based	O
design	O
for	O
other	O
allosteric	O
drug	O
targets	O
including	O
GPCRs	O
and	O
other	O
nuclear	O
receptors	O
.	O
	O
We	O
have	O
solved	O
the	O
structure	O
of	O
the	O
HR1	O
domain	O
of	O
TOCA1	O
,	O
providing	O
the	O
first	O
structural	O
data	O
for	O
this	O
protein	O
.	O
	O
The	O
superfamily	O
can	O
be	O
divided	O
into	O
five	O
families	O
based	O
on	O
structural	O
and	O
functional	O
similarities	O
:	O
Ras	O
,	O
Rho	O
,	O
Rab	O
,	O
Arf	O
,	O
and	O
Ran	O
.	O
	O
These	O
regions	O
are	O
responsible	O
for	O
“	O
sensing	O
”	O
the	O
nucleotide	O
state	O
,	O
with	O
the	O
GTP	O
-	O
bound	O
state	O
showing	O
greater	O
rigidity	O
and	O
the	O
GDP	O
-	O
bound	O
state	O
adopting	O
a	O
more	O
relaxed	O
conformation	O
(	O
reviewed	O
in	O
Ref	O
.).	O
	O
A	O
number	O
of	O
RhoA	O
and	O
Rac1	O
effector	O
proteins	O
,	O
including	O
the	O
formins	O
and	O
members	O
of	O
the	O
protein	O
kinase	O
C	O
-	O
related	O
kinase	O
(	O
PRK	O
)	O
6	O
family	O
,	O
along	O
with	O
Cdc42	O
effectors	O
,	O
including	O
the	O
Wiskott	O
-	O
Aldrich	O
syndrome	O
(	O
WASP	O
)	O
family	O
and	O
the	O
transducer	O
of	O
Cdc42	O
-	O
dependent	O
actin	O
assembly	O
(	O
TOCA	O
)	O
family	O
,	O
have	O
also	O
been	O
linked	O
to	O
the	O
pathways	O
that	O
govern	O
cytoskeletal	O
dynamics	O
.	O
	O
Cdc42	O
effectors	O
,	O
TOCA1	O
and	O
the	O
ubiquitously	O
expressed	O
member	O
of	O
the	O
WASP	O
family	O
,	O
N	O
-	O
WASP	O
,	O
have	O
been	O
implicated	O
in	O
the	O
regulation	O
of	O
actin	O
polymerization	O
downstream	O
of	O
Cdc42	O
and	O
phosphatidylinositol	O
4	O
,	O
5	O
-	O
bisphosphate	O
(	O
PI	O
(	O
4	O
,	O
5	O
)	O
P2	O
).	O
	O
The	O
data	O
were	O
fitted	O
to	O
a	O
binding	O
isotherm	O
to	O
give	O
an	O
apparent	O
Kd	O
and	O
are	O
expressed	O
as	O
a	O
percentage	O
of	O
the	O
maximum	O
signal	O
;	O
B	O
and	O
C	O
,	O
competition	O
SPA	O
experiments	O
were	O
carried	O
out	O
with	O
the	O
indicated	O
concentrations	O
of	O
ACK	O
GBD	O
(	O
B	O
)	O
or	O
HR1	O
domain	O
(	O
C	O
)	O
titrated	O
into	O
30	O
nm	O
GST	B-mutant
-	I-mutant
ACK	I-mutant
and	O
either	O
30	O
nm	O
Cdc42Δ7Q61L	O
·[	O
3H	O
]	O
GTP	O
or	O
full	O
-	O
length	O
Cdc42Q61L	O
·[	O
3H	O
]	O
GTP	O
.	O
	O
The	O
binding	O
experiments	O
were	O
repeated	O
with	O
full	O
-	O
length	O
[	O
3H	O
]	O
GTP	O
·	O
Cdc42	O
,	O
but	O
the	O
affinity	O
of	O
the	O
HR1	O
domain	O
for	O
full	O
-	O
length	O
Cdc42	O
was	O
similar	O
to	O
its	O
affinity	O
for	O
truncated	O
Cdc42	O
(	O
Kd	O
≈	O
5	O
μm	O
;	O
Fig	O
.	O
1C	O
).	O
	O
Another	O
possible	O
explanation	O
for	O
the	O
low	O
affinities	O
observed	O
was	O
that	O
the	O
HR1	O
domain	O
alone	O
is	O
not	O
sufficient	O
for	O
maximal	O
binding	O
of	O
the	O
TOCA	O
proteins	O
to	O
Cdc42	O
and	O
that	O
the	O
other	O
domains	O
are	O
required	O
.	O
	O
Furthermore	O
,	O
both	O
BAR	O
and	O
SH3	O
domains	O
have	O
been	O
implicated	O
in	O
interactions	O
with	O
small	O
G	O
proteins	O
(	O
e	O
.	O
g	O
.	O
the	O
BAR	O
domain	O
of	O
Arfaptin2	O
binds	O
to	O
Rac1	O
and	O
Arl1	O
),	O
while	O
an	O
SH3	O
domain	O
mediates	O
the	O
interaction	O
between	O
Rac1	O
and	O
the	O
guanine	O
nucleotide	O
exchange	O
factor	O
,	O
β	O
-	O
PIX	O
.	O
	O
Full	O
-	O
length	O
TOCA1	O
and	O
ΔSH3	B-mutant
TOCA1	O
bound	O
with	O
micromolar	O
affinity	O
(	O
Fig	O
.	O
2B	O
),	O
in	O
a	O
similar	O
manner	O
to	O
the	O
isolated	O
HR1	O
domain	O
(	O
Fig	O
.	O
1A	O
).	O
	O
There	O
were	O
1	O
,	O
845	O
unambiguous	O
NOEs	O
and	O
757	O
ambiguous	O
NOEs	O
after	O
eight	O
iterations	O
.	O
	O
A	O
sequence	O
alignment	O
illustrating	O
the	O
secondary	O
structure	O
elements	O
of	O
the	O
TOCA1	O
and	O
CIP4	O
HR1	O
domains	O
and	O
the	O
HR1a	O
and	O
HR1b	O
domains	O
from	O
PRK1	O
is	O
shown	O
in	O
Fig	O
.	O
3B	O
.	O
	O
A	O
series	O
of	O
15N	O
HSQC	O
experiments	O
was	O
recorded	O
on	O
15N	O
-	O
labeled	O
TOCA1	O
HR1	O
domain	O
in	O
the	O
presence	O
of	O
increasing	O
concentrations	O
of	O
unlabeled	O
Cdc42Δ7Q61L	O
·	O
GMPPNP	O
to	O
map	O
the	O
Cdc42	O
-	O
binding	O
surface	O
.	O
	O
B	O
,	O
CSPs	O
were	O
calculated	O
as	O
described	O
under	O
“	O
Experimental	O
Procedures	O
”	O
and	O
are	O
shown	O
for	O
backbone	O
and	O
side	O
chain	O
NH	O
groups	O
.	O
	O
Residues	O
that	O
disappeared	O
in	O
the	O
presence	O
of	O
Cdc42	O
were	O
assigned	O
a	O
CSP	O
of	O
0	O
.	O
2	O
but	O
were	O
excluded	O
when	O
calculating	O
the	O
mean	O
CSP	O
and	O
are	O
indicated	O
with	O
open	O
bars	O
.	O
	O
Residues	O
with	O
affected	O
side	O
chain	O
CSPs	O
derived	O
from	O
13C	O
HSQCs	O
are	O
marked	O
with	O
green	O
asterisks	O
above	O
the	O
bars	O
.	O
	O
The	O
corresponding	O
15N	O
and	O
13C	O
NMR	O
experiments	O
were	O
also	O
recorded	O
on	O
15N	O
-	O
Cdc42Δ7Q61L	O
·	O
GMPPNP	O
or	O
15N	O
/	O
13C	O
-	O
Cdc42Δ7Q61L	O
·	O
GMPPNP	O
in	O
the	O
presence	O
of	O
unlabeled	O
HR1	O
domain	O
.	O
	O
A	O
,	O
the	O
15N	O
HSQC	O
of	O
Cdc42Δ7Q61L	O
·	O
GMPPNP	O
is	O
shown	O
in	O
its	O
free	O
form	O
(	O
black	O
)	O
and	O
in	O
the	O
presence	O
of	O
excess	O
TOCA1	O
HR1	O
domain	O
(	O
1	O
:	O
2	O
.	O
2	O
,	O
red	O
).	O
	O
C	O
,	O
the	O
residues	O
with	O
significantly	O
affected	O
backbone	O
and	O
side	O
chain	O
groups	O
are	O
highlighted	O
on	O
an	O
NMR	O
structure	O
of	O
free	O
Cdc42Δ7Q61L	O
·	O
GMPPNP	O
;	O
those	O
that	O
are	O
buried	O
are	O
colored	O
dark	O
blue	O
,	O
whereas	O
those	O
that	O
are	O
solvent	O
-	O
accessible	O
are	O
colored	O
red	O
.	O
	O
Residues	O
without	O
information	O
from	O
shift	O
mapping	O
are	O
colored	O
gray	O
.	O
	O
HADDOCK	O
was	O
therefore	O
used	O
to	O
perform	O
rigid	O
body	O
docking	O
based	O
on	O
the	O
structures	O
of	O
free	O
HR1	O
domain	O
and	O
Cdc42	O
and	O
ambiguous	O
interaction	O
restraints	O
derived	O
from	O
the	O
titration	O
experiments	O
described	O
above	O
.	O
	O
Residues	O
equivalent	O
to	O
Rac1	O
and	O
RhoA	O
contact	O
sites	O
but	O
that	O
are	O
invisible	O
in	O
free	O
Cdc42	O
are	O
gray	O
.	O
	O
D	O
,	O
regions	O
of	O
interest	O
of	O
the	O
Cdc42	O
·	O
HR1	O
domain	O
model	O
.	O
	O
The	O
four	O
lowest	O
energy	O
structures	O
in	O
the	O
chosen	O
HADDOCK	O
cluster	O
are	O
shown	O
overlaid	O
,	O
with	O
the	O
residues	O
of	O
interest	O
shown	O
as	O
sticks	O
and	O
labeled	O
.	O
	O
Lys	O
-	O
16Cdc42	O
is	O
unlikely	O
to	O
be	O
a	O
contact	O
residue	O
because	O
it	O
is	O
involved	O
in	O
nucleotide	O
binding	O
,	O
but	O
the	O
others	O
may	O
represent	O
specific	O
Cdc42	O
-	O
TOCA1	O
contacts	O
.	O
	O
Cdc42	O
is	O
shown	O
in	O
green	O
,	O
and	O
TOCA1	O
is	O
shown	O
in	O
purple	O
.	O
	O
A	O
comparison	O
of	O
the	O
HSQC	O
experiments	O
recorded	O
on	O
15N	O
-	O
Cdc42	O
alone	O
,	O
in	O
the	O
presence	O
of	O
TOCA1	O
HR1	O
,	O
N	O
-	O
WASP	O
GBD	O
,	O
or	O
both	O
,	O
shows	O
that	O
the	O
spectra	O
in	O
the	O
presence	O
of	O
N	O
-	O
WASP	O
and	O
in	O
the	O
presence	O
of	O
both	O
N	O
-	O
WASP	O
and	O
TOCA1	O
HR1	O
are	O
identical	O
(	O
Fig	O
.	O
7C	O
).	O
	O
The	O
spectrum	O
when	O
N	O
-	O
WASP	O
and	O
TOCA1	O
were	O
equimolar	O
was	O
identical	O
to	O
that	O
of	O
the	O
free	O
HR1	O
domain	O
,	O
whereas	O
the	O
spectrum	O
in	O
the	O
presence	O
of	O
0	O
.	O
25	O
eq	O
of	O
N	O
-	O
WASP	O
was	O
intermediate	O
between	O
the	O
TOCA1	O
HR1	O
free	O
and	O
complex	O
spectra	O
(	O
Fig	O
.	O
7D	O
).	O
	O
Taken	O
together	O
,	O
the	O
data	O
in	O
Fig	O
.	O
7	O
,	O
C	O
and	O
D	O
,	O
indicate	O
unidirectional	O
competition	O
for	O
Cdc42	O
binding	O
in	O
which	O
the	O
N	O
-	O
WASP	O
GBD	O
displaces	O
TOCA1	O
HR1	O
but	O
not	O
vice	O
versa	O
.	O
	O
The	O
GBD	O
presumably	O
acts	O
as	O
a	O
dominant	O
negative	O
,	O
sequestering	O
endogenous	O
Cdc42	O
and	O
preventing	O
endogenous	O
full	O
-	O
length	O
N	O
-	O
WASP	O
from	O
binding	O
and	O
becoming	O
activated	O
.	O
	O
The	O
TOCA1	O
HR1	O
domain	O
alone	O
is	O
sufficient	O
for	O
Cdc42	O
binding	O
in	O
vitro	O
,	O
yet	O
the	O
affinity	O
of	O
the	O
TOCA1	O
HR1	O
domain	O
for	O
Cdc42	O
is	O
remarkably	O
low	O
(	O
Kd	O
≈	O
5	O
μm	O
).	O
	O
The	O
polybasic	O
tract	O
within	O
the	O
C	O
-	O
terminal	O
region	O
of	O
Cdc42	O
does	O
not	O
appear	O
to	O
be	O
required	O
for	O
binding	O
to	O
TOCA1	O
,	O
which	O
is	O
in	O
contrast	O
to	O
the	O
interaction	O
between	O
Rac1	O
and	O
the	O
HR1b	O
domain	O
of	O
PRK1	O
but	O
more	O
similar	O
to	O
the	O
PRK1	O
HR1a	O
-	O
RhoA	O
interaction	O
.	O
	O
The	O
equivalent	O
Arg	O
in	O
Rac1	O
and	O
RhoA	O
is	O
pointing	O
away	O
from	O
the	O
HR1	O
domains	O
of	O
PRK1	O
.	O
	O
Furthermore	O
,	O
the	O
isolated	O
F	O
-	O
BAR	O
domain	O
of	O
FBP17	O
has	O
been	O
shown	O
to	O
induce	O
membrane	O
tubulation	O
of	O
brain	O
liposomes	O
and	O
BAR	O
domain	O
proteins	O
that	O
promote	O
tubulation	O
cluster	O
on	O
membranes	O
at	O
high	O
densities	O
.	O
	O
A	O
substantial	O
body	O
of	O
data	O
has	O
illuminated	O
the	O
complex	O
regulation	O
of	O
WASP	O
/	O
N	O
-	O
WASP	O
proteins	O
,	O
and	O
current	O
evidence	O
suggests	O
that	O
these	O
allosteric	O
activation	O
mechanisms	O
and	O
oligomerization	O
combine	O
to	O
regulate	O
WASP	O
activity	O
,	O
allowing	O
the	O
synchronization	O
and	O
integration	O
of	O
multiple	O
potential	O
activation	O
signals	O
(	O
reviewed	O
in	O
Ref	O
.).	O
	O
We	O
envisage	O
that	O
TOCA1	O
is	O
first	O
recruited	O
to	O
the	O
appropriate	O
membrane	O
in	O
response	O
to	O
PI	O
(	O
4	O
,	O
5	O
)	O
P2	O
via	O
its	O
F	O
-	O
BAR	O
domain	O
,	O
where	O
the	O
local	O
increase	O
in	O
concentration	O
favors	O
F	O
-	O
BAR	O
-	O
mediated	O
dimerization	O
of	O
TOCA1	O
.	O
	O
TOCA1	O
can	O
then	O
recruit	O
N	O
-	O
WASP	O
via	O
an	O
interaction	O
between	O
its	O
SH3	O
domain	O
and	O
the	O
N	O
-	O
WASP	O
proline	O
-	O
rich	O
region	O
.	O
	O
In	O
a	O
cellular	O
context	O
,	O
full	O
-	O
length	O
TOCA1	O
and	O
N	O
-	O
WASP	O
are	O
likely	O
to	O
have	O
similar	O
affinities	O
for	O
active	O
Cdc42	O
,	O
but	O
in	O
the	O
unfolded	O
,	O
active	O
conformation	O
,	O
the	O
affinity	O
of	O
N	O
-	O
WASP	O
for	O
Cdc42	O
dramatically	O
increases	O
.	O
	O
Our	O
binding	O
data	O
suggest	O
that	O
TOCA1	O
HR1	O
binding	O
is	O
not	O
allosterically	O
regulated	O
,	O
and	O
our	O
NMR	O
data	O
,	O
along	O
with	O
the	O
high	O
stability	O
of	O
TOCA1	O
HR1	O
,	O
suggest	O
that	O
there	O
is	O
no	O
widespread	O
conformational	O
change	O
in	O
the	O
presence	O
of	O
Cdc42	O
.	O
	O
As	O
full	O
-	O
length	O
TOCA1	O
and	O
the	O
isolated	O
HR1	O
domain	O
bind	O
Cdc42	O
with	O
similar	O
affinities	O
,	O
the	O
N	O
-	O
WASP	O
-	O
Cdc42	O
interaction	O
will	O
be	O
favored	O
because	O
the	O
N	O
-	O
WASP	O
GBD	O
can	O
easily	O
outcompete	O
the	O
TOCA1	O
HR1	O
for	O
Cdc42	O
.	O
	O
Potentially	O
,	O
the	O
TOCA1	O
-	O
Cdc42	O
interaction	O
functions	O
to	O
position	O
N	O
-	O
WASP	O
and	O
Cdc42	O
such	O
that	O
they	O
are	O
poised	O
to	O
interact	O
with	O
high	O
affinity	O
.	O
	O
There	O
is	O
an	O
advantage	O
to	O
such	O
an	O
effector	O
handover	O
,	O
in	O
that	O
N	O
-	O
WASP	O
would	O
only	O
be	O
robustly	O
recruited	O
when	O
F	O
-	O
BAR	O
domains	O
are	O
already	O
present	O
.	O
	O
F	O
-	O
BAR	O
oligomerization	O
is	O
expected	O
to	O
occur	O
following	O
membrane	O
binding	O
,	O
but	O
a	O
single	O
monomer	O
is	O
shown	O
for	O
clarity	O
.	O
	O
The	O
HR1TOCA1	O
-	O
Cdc42	O
and	O
SH3TOCA1	O
-	O
N	O
-	O
WASP	O
interactions	O
position	O
Cdc42	O
and	O
N	O
-	O
WASP	O
for	O
binding	O
.	O
	O
Step	O
4	O
,	O
the	O
core	O
CRIB	O
binds	O
with	O
high	O
affinity	O
while	O
the	O
region	O
C	O
-	O
terminal	O
to	O
the	O
CRIB	O
displaces	O
the	O
TOCA1	O
HR1	O
domain	O
and	O
increases	O
the	O
affinity	O
of	O
the	O
N	O
-	O
WASP	O
-	O
Cdc42	O
interaction	O
further	O
.	O
	O
WH1	O
,	O
WASP	O
homology	O
1	O
domain	O
;	O
PP	O
,	O
proline	O
-	O
rich	O
region	O
;	O
VCA	O
,	O
verprolin	O
homology	O
,	O
cofilin	O
homology	O
,	O
acidic	O
region	O
.	O
	O
We	O
envisage	O
a	O
complex	O
interplay	O
of	O
equilibria	O
between	O
free	O
and	O
bound	O
,	O
active	O
and	O
inactive	O
Cdc42	O
,	O
TOCA	O
family	O
,	O
and	O
WASP	O
family	O
proteins	O
,	O
facilitating	O
a	O
tightly	O
spatially	O
and	O
temporally	O
regulated	O
pathway	O
requiring	O
numerous	O
simultaneous	O
events	O
in	O
order	O
to	O
achieve	O
appropriate	O
and	O
robust	O
activation	O
of	O
the	O
downstream	O
pathway	O
.	O
	O
Acetyl	O
-	O
CoA	O
carboxylases	O
(	O
ACCs	O
)	O
catalyse	O
the	O
committed	O
step	O
in	O
fatty	O
-	O
acid	O
biosynthesis	O
:	O
the	O
ATP	O
-	O
dependent	O
carboxylation	O
of	O
acetyl	O
-	O
CoA	O
to	O
malonyl	O
-	O
CoA	O
.	O
They	O
are	O
important	O
regulatory	O
hubs	O
for	O
metabolic	O
control	O
and	O
relevant	O
drug	O
targets	O
for	O
the	O
treatment	O
of	O
the	O
metabolic	O
syndrome	O
and	O
cancer	O
.	O
	O
Combining	O
the	O
yeast	O
CD	O
structure	O
with	O
intermediate	O
and	O
low	O
-	O
resolution	O
data	O
of	O
larger	B-mutant
fragments	I-mutant
up	O
to	O
intact	O
ACCs	O
provides	O
a	O
comprehensive	O
characterization	O
of	O
the	O
dynamic	O
fungal	O
ACC	O
architecture	O
.	O
	O
In	O
addition	O
to	O
the	O
canonical	O
ACC	O
components	O
,	O
eukaryotic	O
ACCs	O
contain	O
two	O
non	O
-	O
catalytic	O
regions	O
,	O
the	O
large	O
central	O
domain	O
(	O
CD	O
)	O
and	O
the	O
BC	O
–	O
CT	O
interaction	O
domain	O
(	O
BT	O
).	O
	O
The	O
function	O
of	O
this	O
domain	O
remains	O
poorly	O
characterized	O
,	O
although	O
phosphorylation	O
of	O
several	O
serine	O
residues	O
in	O
the	O
CD	O
regulates	O
ACC	O
activity	O
.	O
	O
Of	O
these	O
,	O
only	O
Ser1157	O
is	O
highly	O
conserved	O
in	O
fungal	O
ACC	O
and	O
aligns	O
to	O
Ser1216	O
in	O
human	O
ACC1	O
.	O
	O
Integrating	O
these	O
data	O
with	O
small	O
-	O
angle	O
X	O
-	O
ray	O
scattering	O
(	O
SAXS	O
)	O
and	O
electron	O
microscopy	O
(	O
EM	O
)	O
observations	O
yield	O
a	O
comprehensive	O
representation	O
of	O
the	O
dynamic	O
structure	O
and	O
regulation	O
of	O
fungal	O
ACC	O
.	O
	O
First	O
,	O
we	O
focused	O
on	O
structure	O
determination	O
of	O
the	O
82	O
-	O
kDa	O
CD	O
.	O
	O
Close	O
structural	O
homologues	O
could	O
not	O
be	O
found	O
for	O
the	O
CDN	O
or	O
the	O
CDC	O
domains	O
.	O
	O
To	O
define	O
the	O
functional	O
state	O
of	O
insect	O
-	O
cell	O
-	O
expressed	O
ACC	O
variants	O
,	O
we	O
employed	O
mass	O
spectrometry	O
(	O
MS	O
)	O
for	O
phosphorylation	O
site	O
detection	O
.	O
	O
The	O
SceCD	O
structure	O
thus	O
authentically	O
represents	O
the	O
state	O
of	O
SceACC	O
,	O
where	O
the	O
enzyme	O
is	O
inhibited	O
by	O
SNF1	O
-	O
dependent	O
phosphorylation	O
.	O
	O
Each	O
of	O
the	O
four	O
CD	O
domains	O
in	O
HsaBT	B-mutant
-	I-mutant
CD	I-mutant
individually	O
resembles	O
the	O
corresponding	O
SceCD	O
domain	O
;	O
however	O
,	O
human	O
and	O
yeast	O
CDs	O
exhibit	O
distinct	O
overall	O
structures	O
.	O
	O
In	O
agreement	O
with	O
their	O
tight	O
interaction	O
in	O
SceCD	O
,	O
the	O
relative	O
spatial	O
arrangement	O
of	O
CDL	O
and	O
CDC1	O
is	O
preserved	O
in	O
HsaBT	B-mutant
-	I-mutant
CD	I-mutant
,	O
but	O
the	O
human	O
CDL	O
/	O
CDC1	O
didomain	O
is	O
tilted	O
by	O
30	O
°	O
based	O
on	O
a	O
superposition	O
of	O
human	O
and	O
yeast	O
CDC2	O
(	O
Supplementary	O
Fig	O
.	O
1c	O
).	O
	O
It	O
resembles	O
the	O
BT	O
of	O
propionyl	O
-	O
CoA	O
carboxylase	O
;	O
only	O
the	O
four	O
C	O
-	O
terminal	O
strands	O
of	O
the	O
β	O
-	O
barrel	O
are	O
slightly	O
tilted	O
.	O
	O
The	O
absence	O
of	O
the	O
regulatory	O
loop	O
might	O
be	O
linked	O
to	O
the	O
less	O
-	O
restrained	O
interface	O
of	O
CDL	O
/	O
CDC1	O
and	O
CDC2	O
and	O
altered	O
relative	O
orientations	O
of	O
these	O
domains	O
.	O
	O
To	O
further	O
obtain	O
insights	O
into	O
the	O
functional	O
architecture	O
of	O
fungal	O
ACC	O
,	O
we	O
characterized	O
larger	B-mutant
multidomain	I-mutant
fragments	I-mutant
up	O
to	O
the	O
intact	O
enzymes	O
.	O
	O
No	O
crystals	O
diffracting	O
to	O
sufficient	O
resolution	O
were	O
obtained	O
for	O
larger	B-mutant
BC	I-mutant
-	I-mutant
containing	I-mutant
fragments	I-mutant
,	O
or	O
for	O
full	O
-	O
length	O
Cth	O
or	O
SceACC	O
.	O
	O
However	O
,	O
molecular	O
replacement	O
did	O
not	O
reveal	O
a	O
unique	O
positioning	O
of	O
the	O
BC	O
domain	O
.	O
	O
Indeed	O
,	O
the	O
comparison	O
of	O
the	O
positioning	O
of	O
eight	O
instances	O
of	O
the	O
C	O
-	O
terminal	O
part	O
of	O
CD	O
relative	O
to	O
CT	O
in	O
crystal	O
structures	O
determined	O
here	O
,	O
reveals	O
flexible	O
interdomain	O
linking	O
(	O
Fig	O
.	O
3a	O
).	O
	O
Conformational	O
variability	O
in	O
the	O
CD	O
thus	O
contributes	O
considerably	O
to	O
variations	O
in	O
the	O
spacing	O
between	O
the	O
BC	O
and	O
CT	O
domains	O
,	O
and	O
may	O
extend	O
to	O
distance	O
variations	O
beyond	O
the	O
mobility	O
range	O
of	O
the	O
flexibly	O
tethered	O
BCCP	O
.	O
	O
SAXS	O
analysis	O
of	O
CthACC	O
agrees	O
with	O
a	O
dimeric	O
state	O
and	O
an	O
elongated	O
shape	O
with	O
a	O
maximum	O
extent	O
of	O
350	O
Å	O
(	O
Supplementary	O
Table	O
1	O
).	O
	O
The	O
flexibility	O
in	O
the	O
CDC2	O
/	O
CT	O
hinge	O
appears	O
substantially	O
larger	O
than	O
the	O
variations	O
observed	O
in	O
the	O
set	O
of	O
crystal	O
structures	O
.	O
	O
The	O
phosphorylated	O
regulatory	O
loop	O
binds	O
to	O
an	O
allosteric	O
site	O
at	O
the	O
interface	O
of	O
two	O
non	O
-	O
catalytic	O
domains	O
and	O
restricts	O
conformational	O
freedom	O
at	O
several	O
hinges	O
in	O
the	O
dynamic	O
ACC	O
.	O
	O
(	O
b	O
)	O
Cartoon	O
representation	O
of	O
the	O
SceCD	O
crystal	O
structure	O
.	O
	O
(	O
c	O
)	O
Superposition	O
of	O
CDC1	O
and	O
CDC2	O
reveals	O
highly	O
conserved	O
folds	O
.	O
(	O
d	O
)	O
The	O
regulatory	O
loop	O
with	O
the	O
phosphorylated	O
Ser1157	O
is	O
bound	O
into	O
a	O
crevice	O
between	O
CDC1	O
and	O
CDC2	O
,	O
the	O
conserved	O
residues	O
Arg1173	O
and	O
Arg1260	O
coordinate	O
the	O
phosphoryl	O
-	O
group	O
.	O
	O
The	O
range	O
of	O
hinge	O
bending	O
is	O
indicated	O
and	O
the	O
connection	O
points	O
between	O
CDC2	O
and	O
CT	O
(	O
blue	O
)	O
as	O
well	O
as	O
between	O
CDC1	O
and	O
CDC2	O
(	O
green	O
and	O
grey	O
)	O
are	O
marked	O
as	O
spheres	O
.	O
	O
The	O
connection	O
points	O
from	O
CDC1	O
to	O
CDC2	O
and	O
to	O
CDL	O
are	O
represented	O
by	O
green	O
spheres	O
.	O
	O
The	O
domains	O
are	O
labelled	O
and	O
the	O
distances	O
between	O
the	O
N	O
termini	O
of	O
CDN	O
(	O
spheres	O
)	O
in	O
the	O
compared	O
structures	O
are	O
indicated	O
.	O
	O
The	O
two	O
kinases	O
exhibit	O
nearly	O
identical	O
overall	O
architecture	O
,	O
with	O
both	O
kinases	O
possessing	O
ATP	O
hydrolysis	O
activity	O
in	O
the	O
absence	O
of	O
substrates	O
.	O
	O
SePSK	O
and	O
AtXK	O
-	O
1	O
display	O
a	O
sequence	O
identity	O
of	O
44	O
.	O
9	O
%,	O
and	O
belong	O
to	O
the	O
ribulokinase	O
-	O
like	O
carbohydrate	O
kinases	O
,	O
a	O
sub	O
-	O
family	O
of	O
FGGY	O
family	O
carbohydrate	O
kinases	O
.	O
	O
However	O
,	O
the	O
function	O
of	O
XK	O
-	O
1	O
(	O
At2g21370	O
)	O
inside	O
the	O
chloroplast	O
stroma	O
has	O
remained	O
unknown	O
.	O
	O
Among	O
all	O
these	O
structural	O
elements	O
,	O
α4	O
/	O
α5	O
/	O
α11	O
/	O
α18	O
,	O
β3	O
/	O
β2	O
/	O
β1	O
/	O
β6	O
/	O
β19	O
/	O
β20	O
/	O
β17	O
and	O
α21	O
/	O
α32	O
form	O
three	O
patches	O
,	O
referred	O
to	O
as	O
A1	O
,	O
B1	O
and	O
A2	O
,	O
exhibiting	O
the	O
core	O
region	O
.	O
	O
The	O
structures	O
most	O
closely	O
related	O
to	O
SePSK	O
are	O
xylulose	O
kinase	O
,	O
glycerol	O
kinase	O
and	O
ribulose	O
kinase	O
,	O
implying	O
that	O
SePSK	O
and	O
AtXK	O
-	O
1	O
might	O
function	O
similarly	O
to	O
these	O
kinases	O
.	O
	O
To	O
further	O
identify	O
the	O
actual	O
substrate	O
of	O
SePSK	O
and	O
AtXK	O
-	O
1	O
,	O
five	O
different	O
sugar	O
molecules	O
,	O
including	O
D	O
-	O
ribulose	O
,	O
L	O
-	O
ribulose	O
,	O
D	O
-	O
xylulose	O
,	O
L	O
-	O
xylulose	O
and	O
Glycerol	O
,	O
were	O
used	O
in	O
enzymatic	O
activity	O
assays	O
.	O
	O
While	O
the	O
ATP	O
hydrolysis	O
activity	O
of	O
SePSK	O
greatly	O
increases	O
upon	O
addition	O
of	O
D	O
-	O
ribulose	O
(	O
DR	O
).	O
	O
(	O
B	O
)	O
The	O
ATP	O
hydrolysis	O
activity	O
of	O
SePSK	O
with	O
addition	O
of	O
five	O
different	O
substrates	O
.	O
	O
To	O
obtain	O
more	O
detailed	O
information	O
of	O
SePSK	O
and	O
AtXK	O
-	O
1	O
in	O
complex	O
with	O
ATP	O
,	O
we	O
soaked	O
the	O
apo	O
-	O
crystals	O
in	O
the	O
reservoir	O
adding	O
cofactor	O
ATP	O
,	O
and	O
obtained	O
the	O
structures	O
of	O
SePSK	O
and	O
AtXK	O
-	O
1	O
bound	O
with	O
ATP	O
at	O
the	O
resolution	O
of	O
2	O
.	O
3	O
Å	O
and	O
1	O
.	O
8	O
Å	O
,	O
respectively	O
.	O
	O
Thus	O
the	O
two	O
structures	O
were	O
named	O
ADP	O
-	O
SePSK	O
and	O
ADP	O
-	O
AtXK	O
-	O
1	O
,	O
respectively	O
.	O
	O
Structure	O
of	O
SePSK	O
in	O
complex	O
with	O
AMP	O
-	O
PNP	O
.	O
	O
(	O
A	O
)	O
The	O
electron	O
density	O
of	O
AMP	O
-	O
PNP	O
.	O
	O
The	O
AMP	O
-	O
PNP	O
is	O
depicted	O
as	O
sticks	O
with	O
its	O
ǀFoǀ	O
-	O
ǀFcǀ	O
map	O
contoured	O
at	O
3	O
σ	O
shown	O
as	O
cyan	O
mesh	O
.	O
	O
(	O
B	O
)	O
The	O
AMP	O
-	O
PNP	O
binding	O
pocket	O
.	O
	O
The	O
AMP	O
-	O
PNP	O
and	O
coordinated	O
residues	O
are	O
shown	O
as	O
sticks	O
.	O
	O
The	O
potential	O
substrate	O
binding	O
site	O
in	O
SePSK	O
	O
The	O
RBL1	O
and	O
RBL2	O
are	O
depicted	O
as	O
sticks	O
.	O
(	O
B	O
)	O
Interaction	O
of	O
two	O
D	O
-	O
ribulose	O
molecules	O
(	O
RBL1	O
and	O
RBL2	O
)	O
with	O
SePSK	O
.	O
	O
The	O
RBL	O
molecules	O
(	O
carbon	O
atoms	O
colored	O
yellow	O
)	O
and	O
amino	O
acid	O
residues	O
of	O
SePSK	O
(	O
carbon	O
atoms	O
colored	O
green	O
)	O
involved	O
in	O
RBL	O
interaction	O
are	O
shown	O
as	O
sticks	O
.	O
	O
The	O
hydroxyl	O
group	O
of	O
Ser12	O
coordinates	O
with	O
O2	O
of	O
RBL2	O
.	O
	O
Structural	O
comparison	O
of	O
SePSK	O
and	O
AtXK	O
-	O
1	O
showed	O
that	O
while	O
the	O
RBL1	O
binding	O
pocket	O
is	O
conserved	O
,	O
the	O
RBL2	O
pocket	O
is	O
disrupted	O
in	O
AtXK	O
-	O
1	O
structure	O
,	O
despite	O
the	O
fact	O
that	O
the	O
residues	O
interacting	O
with	O
RBL2	O
are	O
highly	O
conserved	O
between	O
the	O
two	O
proteins	O
.	O
	O
In	O
the	O
RBL	O
-	O
SePSK	O
structure	O
,	O
a	O
2	O
.	O
6	O
Å	O
hydrogen	O
bond	O
is	O
present	O
between	O
RBL2	O
and	O
Ser12	O
(	O
Fig	O
4B	O
),	O
while	O
in	O
the	O
AtXK	O
-	O
1	O
structure	O
this	O
hydrogen	O
bond	O
with	O
the	O
corresponding	O
residue	O
(	O
Ser22	O
)	O
is	O
broken	O
.	O
	O
This	O
change	O
might	O
be	O
the	O
reason	O
that	O
AtXK	O
-	O
1	O
only	O
shows	O
limited	O
increasing	O
in	O
its	O
ATP	O
hydrolysis	O
ability	O
upon	O
adding	O
D	O
-	O
ribulose	O
as	O
a	O
substrate	O
after	O
comparing	O
with	O
SePSK	O
(	O
Fig	O
2C	O
).	O
	O
The	O
results	O
showed	O
that	O
the	O
affinity	O
of	O
D8A	B-mutant
-	O
SePSK	O
with	O
D	O
-	O
ribulose	O
is	O
weaker	O
than	O
that	O
of	O
WT	O
with	O
a	O
reduction	O
of	O
approx	O
.	O
	O
This	O
distance	O
between	O
RBL2	O
and	O
AMP	O
-	O
PNP	O
-	O
γ	O
-	O
phosphate	O
is	O
close	O
enough	O
to	O
facilitate	O
phosphate	O
transferring	O
.	O
	O
Together	O
,	O
our	O
superposition	O
results	O
provided	O
snapshots	O
of	O
the	O
conformational	O
changes	O
at	O
different	O
catalytic	O
stages	O
of	O
SePSK	O
and	O
potentially	O
revealed	O
the	O
closed	O
form	O
of	O
SePSK	O
.	O
	O
In	O
summary	O
,	O
our	O
structural	O
and	O
enzymatic	O
analyses	O
provide	O
evidence	O
that	O
SePSK	O
shows	O
D	O
-	O
ribulose	O
kinase	O
activity	O
,	O
and	O
exhibits	O
the	O
conserved	O
features	O
of	O
FGGY	O
family	O
carbohydrate	O
kinases	O
.	O
	O
Three	O
conserved	O
residues	O
in	O
SePSK	O
were	O
identified	O
to	O
be	O
essential	O
for	O
this	O
function	O
.	O
	O
We	O
now	O
present	O
cryo	O
-	O
electron	O
microscopy	O
3D	O
reconstructions	O
of	O
the	O
E	O
.	O
coli	O
LdcI	O
and	O
LdcC	O
,	O
and	O
an	O
improved	O
map	O
of	O
the	O
LdcI	O
bound	O
to	O
the	O
LARA	O
domain	O
of	O
RavA	O
,	O
at	O
pH	O
optimal	O
for	O
their	O
enzymatic	O
activity	O
.	O
	O
They	O
counteract	O
acid	O
stress	O
experienced	O
by	O
the	O
bacterium	O
in	O
the	O
host	O
digestive	O
and	O
urinary	O
tract	O
,	O
and	O
in	O
particular	O
in	O
the	O
extremely	O
acidic	O
stomach	O
.	O
	O
Monomers	O
tightly	O
associate	O
via	O
their	O
core	O
domains	O
into	O
2	O
-	O
fold	O
symmetrical	O
dimers	O
with	O
two	O
complete	O
active	O
sites	O
,	O
and	O
further	O
build	O
a	O
toroidal	O
D5	O
-	O
symmetrical	O
structure	O
held	O
by	O
the	O
wing	O
and	O
core	O
domain	O
interactions	O
around	O
the	O
central	O
pore	O
,	O
with	O
the	O
CTDs	O
at	O
the	O
periphery	O
.	O
	O
This	O
allowed	O
us	O
to	O
make	O
a	O
pseudoatomic	O
model	O
of	O
the	O
whole	O
assembly	O
,	O
underpinned	O
by	O
a	O
cryoEM	O
map	O
of	O
the	O
LdcI	O
-	O
LARA	O
complex	O
(	O
with	O
LARA	O
standing	O
for	O
LdcI	O
associating	O
domain	O
of	O
RavA	O
),	O
and	O
to	O
identify	O
conformational	O
rearrangements	O
and	O
specific	O
elements	O
essential	O
for	O
complex	O
formation	O
.	O
	O
The	O
main	O
determinants	O
of	O
the	O
LdcI	O
-	O
RavA	O
cage	O
assembly	O
appeared	O
to	O
be	O
the	O
N	O
-	O
terminal	O
loop	O
of	O
the	O
LARA	O
domain	O
of	O
RavA	O
and	O
the	O
C	O
-	O
terminal	O
β	O
-	O
sheet	O
of	O
LdcI	O
.	O
	O
Finally	O
,	O
we	O
performed	O
multiple	O
sequence	O
alignment	O
of	O
22	O
lysine	O
decarboxylases	O
from	O
Enterobacteriaceae	O
containing	O
the	O
ravA	O
-	O
viaA	O
operon	O
in	O
their	O
genome	O
.	O
	O
Significant	O
differences	O
between	O
these	O
pseudoatomic	O
models	O
can	O
be	O
interpreted	O
as	O
movements	O
between	O
specific	O
biological	O
states	O
of	O
the	O
proteins	O
as	O
described	O
below	O
.	O
	O
Both	O
visual	O
inspection	O
(	O
Fig	O
.	O
2	O
)	O
and	O
RMSD	O
calculations	O
(	O
Table	O
S2	O
)	O
show	O
that	O
globally	O
the	O
three	O
structures	O
at	O
active	O
pH	O
(	O
LdcIa	O
,	O
LdcI	O
-	O
LARA	O
and	O
LdcC	O
)	O
are	O
more	O
similar	O
to	O
each	O
other	O
than	O
to	O
the	O
structure	O
determined	O
at	O
high	O
pH	O
conditions	O
(	O
LdcIi	O
).	O
	O
The	O
core	O
domain	O
is	O
built	O
by	O
the	O
PLP	O
-	O
binding	O
subdomain	O
(	O
PLP	O
-	O
SD	O
,	O
residues	O
184	O
–	O
417	O
)	O
flanked	O
by	O
two	O
smaller	O
subdomains	O
rich	O
in	O
partly	O
disordered	O
loops	O
–	O
the	O
linker	O
region	O
(	O
residues	O
130	O
–	O
183	O
)	O
and	O
the	O
subdomain	O
4	O
(	O
residues	O
418	O
–	O
563	O
).	O
	O
In	O
particular	O
,	O
transition	O
from	O
LdcIi	O
to	O
LdcI	O
-	O
LARA	O
involves	O
~	O
3	O
.	O
5	O
Å	O
and	O
~	O
4	O
.	O
5	O
Å	O
shifts	O
away	O
from	O
the	O
5	O
-	O
fold	O
axis	O
in	O
the	O
active	O
site	O
α	O
-	O
helices	O
spanning	O
residues	O
218	O
–	O
232	O
and	O
246	O
–	O
254	O
respectively	O
(	O
Fig	O
.	O
3C	O
–	O
E	O
).	O
	O
At	O
this	O
resolution	O
,	O
the	O
apo	O
-	O
LdcIi	O
and	O
ppGpp	O
-	O
LdcIi	O
structures	O
(	O
both	O
solved	O
at	O
pH	O
8	O
.	O
5	O
)	O
appeared	O
indistinguishable	O
except	O
for	O
the	O
presence	O
of	O
ppGpp	O
(	O
Fig	O
.	O
S11	O
in	O
ref	O
.	O
).	O
	O
Yet	O
the	O
superposition	O
of	O
the	O
decamers	O
lays	O
bare	O
a	O
progressive	O
movement	O
of	O
the	O
CTD	O
as	O
a	O
whole	O
upon	O
enzyme	O
activation	O
by	O
pH	O
and	O
the	O
binding	O
of	O
LARA	O
.	O
	O
On	O
the	O
contrary	O
,	O
introduction	O
of	O
the	O
C	O
-	O
terminal	O
β	O
-	O
sheet	O
of	O
LdcI	O
into	O
LdcC	O
led	O
to	O
an	O
assembly	O
of	O
the	O
LdcCI	O
-	O
RavA	O
complex	O
.	O
	O
(	O
A	O
,	O
C	O
,	O
E	O
)	O
cryoEM	O
map	O
of	O
the	O
LdcC	O
(	O
A	O
),	O
LdcIa	O
(	O
C	O
)	O
and	O
LdcI	O
-	O
LARA	O
(	O
E	O
)	O
decamers	O
with	O
one	O
protomer	O
in	O
light	O
grey	O
.	O
	O
Only	O
one	O
of	O
the	O
two	O
rings	O
of	O
the	O
double	O
toroid	O
is	O
shown	O
for	O
clarity	O
.	O
	O
Conformational	O
rearrangements	O
in	O
the	O
enzyme	O
active	O
site	O
.	O
	O
(	O
A	O
)	O
A	O
slice	O
through	O
the	O
pseudoatomic	O
models	O
of	O
the	O
LdcIa	O
(	O
purple	O
)	O
and	O
LdcC	O
(	O
green	O
)	O
monomers	O
extracted	O
from	O
the	O
superimposed	O
decamers	O
(	O
Fig	O
.	O
2	O
).	O
(	O
B	O
)	O
The	O
C	O
-	O
terminal	O
β	O
-	O
sheet	O
in	O
LdcIa	O
and	O
LdcC	O
enlarged	O
from	O
(	O
A	O
,	O
C	O
)	O
Exchanged	O
primary	O
sequences	O
(	O
capital	O
letters	O
)	O
and	O
their	O
immediate	O
vicinity	O
(	O
lower	O
case	O
letters	O
)	O
colored	O
as	O
in	O
(	O
A	O
,	O
B	O
),	O
with	O
the	O
corresponding	O
secondary	O
structure	O
elements	O
and	O
the	O
amino	O
acid	O
numbering	O
shown	O
.	O
	O
(	O
A	O
)	O
Maximum	O
likelihood	O
tree	O
with	O
the	O
“	O
LdcC	O
-	O
like	O
”	O
and	O
the	O
“	O
LdcI	O
-	O
like	O
”	O
groups	O
highlighted	O
in	O
green	O
and	O
pink	O
,	O
respectively	O
.	O
	O
Structural	O
basis	O
for	O
Mep2	O
ammonium	O
transceptor	O
activation	O
by	O
phosphorylation	O
	O
Mep2	O
proteins	O
are	O
fungal	O
transceptors	O
that	O
play	O
an	O
important	O
role	O
as	O
ammonium	O
sensors	O
in	O
fungal	O
development	O
.	O
	O
While	O
most	O
studies	O
have	O
focused	O
on	O
the	O
Saccharomyces	O
cerevisiae	O
transceptors	O
for	O
phosphate	O
(	O
Pho84	O
),	O
amino	O
acids	O
(	O
Gap1	O
)	O
and	O
ammonium	O
(	O
Mep2	O
),	O
transceptors	O
are	O
found	O
in	O
higher	O
eukaryotes	O
as	O
well	O
(	O
for	O
example	O
,	O
the	O
mammalian	O
SNAT2	O
amino	O
-	O
acid	O
transporter	O
and	O
the	O
GLUT2	O
glucose	O
transporter	O
).	O
	O
Of	O
these	O
,	O
only	O
Mep2	O
proteins	O
function	O
as	O
ammonium	O
receptors	O
/	O
sensors	O
in	O
fungal	O
development	O
.	O
	O
In	O
bacteria	O
,	O
amt	O
genes	O
are	O
present	O
in	O
an	O
operon	O
with	O
glnK	O
,	O
encoding	O
a	O
PII	O
-	O
like	O
signal	O
transduction	O
class	O
protein	O
.	O
	O
Under	O
conditions	O
of	O
nitrogen	O
limitation	O
,	O
GlnK	O
becomes	O
uridylated	O
,	O
blocking	O
its	O
ability	O
to	O
bind	O
and	O
inhibit	O
Amt	O
proteins	O
.	O
	O
(	O
root	O
mean	O
square	O
deviation	O
)=	O
0	O
.	O
7	O
Å	O
for	O
434	O
residues	O
),	O
with	O
the	O
main	O
differences	O
confined	O
to	O
the	O
N	O
terminus	O
and	O
the	O
CTR	O
(	O
Fig	O
.	O
1	O
).	O
	O
The	O
N	O
termini	O
of	O
the	O
Mep2	O
proteins	O
are	O
∼	O
20	O
–	O
25	O
residues	O
longer	O
compared	O
with	O
their	O
bacterial	O
counterparts	O
(	O
Figs	O
1	O
and	O
2	O
),	O
substantially	O
increasing	O
the	O
size	O
of	O
the	O
extracellular	O
domain	O
.	O
	O
The	O
N	O
-	O
terminal	O
vestibule	O
and	O
the	O
resulting	O
inter	O
-	O
monomer	O
interactions	O
likely	O
increase	O
the	O
stability	O
of	O
the	O
Mep2	O
trimer	O
,	O
in	O
support	O
of	O
data	O
for	O
plant	O
AMT	O
proteins	O
.	O
	O
The	O
head	O
group	O
of	O
Arg54	O
has	O
moved	O
∼	O
11	O
Å	O
relative	O
to	O
that	O
in	O
Amt	O
-	O
1	O
,	O
whereas	O
the	O
shift	O
of	O
the	O
head	O
group	O
of	O
the	O
variable	O
Lys55	O
residue	O
is	O
almost	O
20	O
Å	O
.	O
The	O
side	O
chain	O
of	O
Lys56	O
in	O
the	O
basic	O
motif	O
points	O
in	O
an	O
opposite	O
direction	O
in	O
the	O
Mep2	O
structures	O
compared	O
with	O
that	O
of	O
,	O
for	O
example	O
,	O
Amt	O
-	O
1	O
(	O
Fig	O
.	O
4	O
).	O
	O
Significantly	O
,	O
this	O
is	O
also	O
true	O
for	O
ScMep2	O
,	O
which	O
was	O
crystallized	O
in	O
the	O
presence	O
of	O
0	O
.	O
2	O
M	O
ammonium	O
ions	O
(	O
see	O
Methods	O
section	O
).	O
	O
In	O
Mep2	O
,	O
the	O
CTR	O
has	O
moved	O
away	O
and	O
makes	O
relatively	O
few	O
contacts	O
with	O
the	O
main	O
body	O
of	O
the	O
transporter	O
,	O
generating	O
a	O
more	O
elongated	O
protein	O
(	O
Figs	O
1	O
and	O
4	O
).	O
	O
These	O
residues	O
include	O
those	O
of	O
the	O
‘	O
ExxGxD	O
'	O
motif	O
,	O
which	O
when	O
mutated	O
generate	O
inactive	O
transporters	O
.	O
	O
In	O
Amt	O
-	O
1	O
and	O
other	O
bacterial	O
ammonium	O
transporters	O
,	O
these	O
CTR	O
residues	O
interact	O
with	O
residues	O
within	O
the	O
N	O
-	O
terminal	O
half	O
of	O
the	O
protein	O
.	O
	O
At	O
the	O
other	O
end	O
of	O
ICL3	O
,	O
the	O
backbone	O
carbonyl	O
groups	O
of	O
Gly172	O
and	O
Lys173	O
are	O
hydrogen	O
bonded	O
to	O
the	O
side	O
chain	O
of	O
Arg370	O
.	O
	O
This	O
interaction	O
in	O
the	O
centre	O
of	O
the	O
protein	O
may	O
be	O
particularly	O
important	O
to	O
stabilize	O
the	O
open	O
conformations	O
of	O
ammonium	O
transporters	O
.	O
	O
Where	O
is	O
the	O
AI	O
region	O
and	O
the	O
Npr1	O
phosphorylation	O
site	O
located	O
?	O
Our	O
structures	O
reveal	O
that	O
surprisingly	O
,	O
the	O
AI	O
region	O
is	O
folded	O
back	O
onto	O
the	O
CTR	O
and	O
is	O
not	O
located	O
near	O
the	O
centre	O
of	O
the	O
trimer	O
as	O
expected	O
from	O
the	O
bacterial	O
structures	O
(	O
Fig	O
.	O
4	O
).	O
	O
The	O
AI	O
regions	O
have	O
very	O
similar	O
conformations	O
in	O
CaMep2	O
and	O
ScMep2	O
,	O
despite	O
considerable	O
differences	O
in	O
the	O
rest	O
of	O
the	O
CTR	O
(	O
Fig	O
.	O
6	O
).	O
	O
This	O
makes	O
sense	O
since	O
the	O
proteins	O
were	O
expressed	O
in	O
rich	O
medium	O
and	O
confirms	O
the	O
recent	O
suggestion	O
by	O
Boeckstaens	O
et	O
al	O
.	O
that	O
the	O
non	O
-	O
phosphorylated	O
form	O
of	O
Mep2	O
corresponds	O
to	O
the	O
inactive	O
state	O
.	O
	O
The	O
peripheral	O
location	O
and	O
disorder	O
of	O
the	O
CTR	O
beyond	O
the	O
kinase	O
target	O
site	O
should	O
facilitate	O
the	O
phosphorylation	O
by	O
Npr1	O
.	O
	O
Mep2	O
lacking	O
the	O
AI	O
region	O
is	O
conformationally	O
heterogeneous	O
	O
Density	O
for	O
ICL3	O
and	O
the	O
CTR	O
beyond	O
residue	O
Arg415	O
is	O
missing	O
in	O
the	O
442Δ	B-mutant
mutant	O
,	O
and	O
the	O
density	O
for	O
the	O
other	O
ICLs	O
including	O
ICL1	O
is	O
generally	O
poor	O
with	O
visible	O
parts	O
of	O
the	O
structure	O
having	O
high	O
B	O
-	O
factors	O
(	O
Fig	O
.	O
7	O
).	O
	O
Why	O
then	O
does	O
this	O
mutant	O
appear	O
to	O
be	O
constitutively	O
active	O
?	O
We	O
propose	O
two	O
possibilities	O
.	O
	O
The	O
latter	O
model	O
would	O
fit	O
well	O
with	O
the	O
NH3	O
/	O
H	O
+	O
symport	O
model	O
in	O
which	O
the	O
proton	O
is	O
relayed	O
by	O
the	O
twin	O
-	O
His	O
motif	O
.	O
	O
To	O
test	O
this	O
hypothesis	O
,	O
we	O
determined	O
the	O
structure	O
of	O
the	O
phosphorylation	O
-	O
mimicking	O
R452D	B-mutant
/	I-mutant
S453D	I-mutant
protein	O
(	O
hereafter	O
termed	O
‘	O
DD	B-mutant
mutant	I-mutant
'),	O
using	O
data	O
to	O
a	O
resolution	O
of	O
2	O
.	O
4	O
Å	O
.	O
The	O
additional	O
mutation	O
of	O
the	O
arginine	O
preceding	O
the	O
phosphorylation	O
site	O
was	O
introduced	O
(	O
i	O
)	O
to	O
increase	O
the	O
negative	O
charge	O
density	O
and	O
make	O
it	O
more	O
comparable	O
to	O
a	O
phosphate	O
at	O
neutral	O
pH	O
,	O
and	O
(	O
ii	O
)	O
to	O
further	O
destabilize	O
the	O
interactions	O
of	O
the	O
AI	O
region	O
with	O
the	O
main	O
body	O
of	O
the	O
transporter	O
(	O
Fig	O
.	O
6	O
).	O
	O
In	O
addition	O
,	O
residues	O
Glu420	O
-	O
Leu423	O
including	O
Glu421	O
of	O
the	O
ExxGxD	O
motif	O
are	O
now	O
disordered	O
(	O
Fig	O
.	O
8	O
and	O
Supplementary	O
Fig	O
.	O
3	O
).	O
	O
The	O
protein	O
backbone	O
has	O
an	O
average	O
r	O
.	O
m	O
.	O
s	O
.	O
d	O
.	O
of	O
only	O
∼	O
3	O
Å	O
during	O
the	O
200	O
-	O
ns	O
simulation	O
,	O
indicating	O
that	O
the	O
protein	O
is	O
stable	O
.	O
	O
There	O
is	O
flexibility	O
in	O
the	O
side	O
chains	O
of	O
the	O
acidic	O
residues	O
so	O
that	O
they	O
are	O
able	O
to	O
form	O
stable	O
hydrogen	O
bonds	O
with	O
Ser453	O
.	O
	O
For	O
example	O
,	O
the	O
distance	O
between	O
the	O
Asp453	O
acidic	O
oxygens	O
and	O
the	O
Glu420	O
acidic	O
oxygens	O
increases	O
from	O
∼	O
7	O
to	O
>	O
22	O
Å	O
after	O
200	O
ns	O
simulations	O
,	O
and	O
thus	O
these	O
residues	O
are	O
not	O
interacting	O
.	O
	O
The	O
distance	O
between	O
the	O
phosphate	O
of	O
Sep453	O
and	O
the	O
acidic	O
oxygen	O
atoms	O
of	O
Glu420	O
is	O
initially	O
∼	O
11	O
Å	O
,	O
but	O
increases	O
to	O
>	O
30	O
Å	O
after	O
200	O
ns	O
.	O
	O
More	O
specifically	O
,	O
the	O
close	O
interactions	O
between	O
the	O
CTR	O
and	O
ICL1	O
/	O
ICL3	O
present	O
in	O
open	O
transporters	O
are	O
disrupted	O
,	O
causing	O
ICL3	O
to	O
move	O
outwards	O
and	O
block	O
the	O
channel	O
(	O
Figs	O
4	O
and	O
9a	O
).	O
	O
However	O
,	O
even	O
the	O
otherwise	O
highly	O
similar	O
Mep2	O
proteins	O
of	O
S	O
.	O
cerevisiae	O
and	O
C	O
.	O
albicans	O
have	O
different	O
structures	O
for	O
their	O
CTRs	O
(	O
Fig	O
.	O
1	O
and	O
Supplementary	O
Fig	O
.	O
6	O
).	O
	O
In	O
addition	O
,	O
the	O
considerable	O
differences	O
between	O
structurally	O
resolved	O
CTR	O
domains	O
means	O
that	O
the	O
exact	O
environment	O
of	O
T460	O
in	O
Amt	O
-	O
1	O
;	O
1	O
is	O
also	O
not	O
known	O
(	O
Supplementary	O
Fig	O
.	O
6	O
).	O
	O
(	O
a	O
)	O
The	O
triple	B-mutant
mepΔ	I-mutant
strain	O
(	O
black	O
)	O
and	O
triple	O
mepΔ	O
npr1Δ	O
strain	O
(	O
grey	O
)	O
containing	O
plasmids	O
expressing	O
WT	O
and	O
variant	B-mutant
ScMep2	I-mutant
were	O
grown	O
on	O
minimal	O
medium	O
containing	O
1	O
mM	O
ammonium	O
sulphate	O
.	O
	O
The	O
numbering	O
is	O
for	O
CaMep2	O
.	O
	O
Channel	O
closures	O
in	O
Mep2	O
.	O
	O
2Fo	O
–	O
Fc	O
electron	O
density	O
(	O
contoured	O
at	O
1	O
.	O
0	O
σ	O
)	O
for	O
residues	O
Tyr49	O
and	O
His342	O
is	O
shown	O
for	O
the	O
truncation	O
mutant	O
.	O
	O
The	O
arrow	O
indicates	O
the	O
phosphorylation	O
site	O
.	O
	O
Upon	O
phosphorylation	O
and	O
mimicked	O
by	O
the	O
CaMep2	O
S453D	B-mutant
and	O
DD	B-mutant
mutants	I-mutant
(	O
ii	O
),	O
the	O
region	O
around	O
the	O
ExxGxD	O
motif	O
undergoes	O
a	O
conformational	O
change	O
that	O
results	O
in	O
the	O
CTR	O
interacting	O
with	O
the	O
inward	O
-	O
moving	O
ICL3	O
,	O
opening	O
the	O
channel	O
(	O
full	O
circle	O
)	O
(	O
iii	O
).	O
	O
Once	O
a	O
candidate	O
antibody	O
is	O
identified	O
,	O
protein	O
engineering	O
is	O
usually	O
required	O
to	O
produce	O
a	O
molecule	O
with	O
the	O
right	O
biophysical	O
and	O
functional	O
properties	O
.	O
	O
The	O
sequence	O
diversity	O
of	O
the	O
CDR	O
regions	O
presents	O
a	O
substantial	O
challenge	O
to	O
antibody	O
modeling	O
.	O
	O
In	O
contrast	O
to	O
CDRs	O
L1	O
,	O
L2	O
,	O
L3	O
,	O
H1	O
and	O
H2	O
,	O
no	O
canonical	O
structures	O
have	O
been	O
observed	O
for	O
CDR	O
H3	O
,	O
which	O
is	O
the	O
most	O
variable	O
in	O
length	O
and	O
amino	O
acid	O
sequence	O
.	O
	O
Some	O
clustering	O
of	O
conformations	O
was	O
observed	O
for	O
the	O
shortest	O
lengths	O
;	O
however	O
,	O
for	O
the	O
longer	O
loops	O
,	O
only	O
the	O
portions	O
nearest	O
the	O
framework	O
(	O
torso	O
,	O
stem	O
or	O
anchor	O
region	O
)	O
were	O
found	O
to	O
have	O
defined	O
conformations	O
.	O
	O
Current	O
antibody	O
modeling	O
approaches	O
take	O
advantage	O
of	O
the	O
most	O
recent	O
advances	O
in	O
homology	O
modeling	O
,	O
the	O
evolving	O
understanding	O
of	O
the	O
CDR	O
canonical	O
structures	O
,	O
the	O
emerging	O
rules	O
for	O
CDR	O
H3	O
modeling	O
and	O
the	O
growing	O
body	O
of	O
antibody	O
structural	O
data	O
available	O
from	O
the	O
PDB	O
.	O
	O
To	O
support	O
antibody	O
engineering	O
and	O
therapeutic	O
development	O
efforts	O
,	O
a	O
phage	O
library	O
was	O
designed	O
and	O
constructed	O
based	O
on	O
a	O
limited	O
number	O
of	O
scaffolds	O
built	O
with	O
frequently	O
used	O
human	O
germ	O
-	O
line	O
IGV	O
and	O
IGJ	O
gene	O
segments	O
that	O
encode	O
antigen	O
combining	O
sites	O
suitable	O
for	O
recognition	O
of	O
peptides	O
and	O
proteins	O
.	O
	O
Variations	O
occur	O
in	O
the	O
pH	O
(	O
buffer	O
)	O
and	O
the	O
additives	O
,	O
and	O
,	O
in	O
group	O
3	O
,	O
PEG	O
3350	O
is	O
the	O
precipitant	O
for	O
one	O
variants	O
while	O
ammonium	O
sulfate	O
is	O
the	O
precipitant	O
for	O
the	O
other	O
two	O
.	O
	O
Apart	O
from	O
the	O
C	O
-	O
terminus	O
,	O
only	O
a	O
few	O
surface	O
residues	O
in	O
LC	O
are	O
disordered	O
.	O
	O
The	O
HCs	O
feature	O
the	O
largest	O
number	O
of	O
disordered	O
residues	O
,	O
with	O
the	O
lower	O
resolution	O
structures	O
having	O
the	O
most	O
.	O
	O
CDR	O
H1	O
and	O
CDR	O
H2	O
also	O
show	O
some	O
degree	O
of	O
disorder	O
,	O
but	O
to	O
a	O
lesser	O
extent	O
.	O
	O
Three	O
of	O
the	O
HCs	O
,	O
H3	B-mutant
-	I-mutant
23	I-mutant
,	O
H3	B-mutant
-	I-mutant
53	I-mutant
and	O
H5	B-mutant
-	I-mutant
51	I-mutant
,	O
have	O
the	O
same	O
canonical	O
structure	O
,	O
H1	B-mutant
-	I-mutant
13	I-mutant
-	I-mutant
1	I-mutant
,	O
and	O
the	O
backbone	O
conformations	O
are	O
tightly	O
clustered	O
for	O
each	O
set	O
of	O
Fab	O
structures	O
as	O
reflected	O
in	O
the	O
rmsd	O
values	O
(	O
Fig	O
.	O
1B	O
-	O
D	O
).	O
	O
Each	O
of	O
the	O
4	O
HCs	O
adopts	O
only	O
one	O
canonical	O
structure	O
regardless	O
of	O
the	O
pairing	O
LC	O
.	O
	O
Germlines	O
H1	B-mutant
-	I-mutant
69	I-mutant
and	O
H5	B-mutant
-	I-mutant
51	I-mutant
have	O
the	O
same	O
canonical	O
structure	O
assignment	O
H2	B-mutant
-	I-mutant
10	I-mutant
-	I-mutant
1	I-mutant
,	O
H3	B-mutant
-	I-mutant
23	I-mutant
has	O
H2	B-mutant
-	I-mutant
10	I-mutant
-	I-mutant
2	I-mutant
,	O
and	O
H3	B-mutant
-	I-mutant
53	I-mutant
has	O
H2	B-mutant
-	I-mutant
9	I-mutant
-	I-mutant
3	I-mutant
.	O
	O
Germlines	O
H1	B-mutant
-	I-mutant
69	I-mutant
and	O
H5	B-mutant
-	I-mutant
51	I-mutant
are	O
unique	O
in	O
the	O
human	O
repertoire	O
in	O
having	O
an	O
Ala	O
at	O
position	O
71	O
that	O
leaves	O
enough	O
space	O
for	O
H	O
-	O
Pro52a	O
to	O
pack	O
deeper	O
against	O
CDR	O
H4	O
so	O
that	O
the	O
following	O
residues	O
53	O
and	O
54	O
point	O
toward	O
the	O
putative	O
antigen	O
.	O
	O
However	O
,	O
there	O
is	O
a	O
significant	O
shift	O
of	O
the	O
CDR	O
as	O
a	O
rigid	O
body	O
when	O
the	O
2	O
sets	O
are	O
superimposed	O
.	O
	O
Germline	O
H1	B-mutant
-	I-mutant
69	I-mutant
has	O
Ala	O
at	O
position	O
33	O
whereas	O
in	O
H5	B-mutant
-	I-mutant
51	I-mutant
position	O
33	O
is	O
occupied	O
by	O
a	O
bulky	O
Trp	O
,	O
which	O
stacks	O
against	O
H	O
-	O
Tyr52	O
and	O
drives	O
CDR	O
H2	O
away	O
from	O
the	O
center	O
.	O
	O
For	O
the	O
remaining	O
2	O
,	O
L3	B-mutant
-	I-mutant
20	I-mutant
has	O
2	O
different	O
assignments	O
,	O
L1	B-mutant
-	I-mutant
12	I-mutant
-	I-mutant
1	I-mutant
and	O
L1	B-mutant
-	I-mutant
12	I-mutant
-	I-mutant
2	I-mutant
,	O
while	O
L4	B-mutant
-	I-mutant
1	I-mutant
has	O
a	O
single	O
assignment	O
,	O
L1	B-mutant
-	I-mutant
17	I-mutant
-	I-mutant
1	I-mutant
.	O
	O
L3	B-mutant
-	I-mutant
20	I-mutant
is	O
the	O
most	O
variable	O
in	O
CDR	O
L1	O
among	O
the	O
4	O
germlines	O
as	O
indicated	O
by	O
an	O
rmsd	O
of	O
0	O
.	O
54	O
Å	O
(	O
Fig	O
.	O
3C	O
).	O
	O
The	O
third	O
structure	O
,	O
H3	O
-	O
23	O
:	O
L3	O
-	O
20	O
,	O
has	O
CDR	O
L1	O
as	O
L1	B-mutant
-	I-mutant
12	I-mutant
-	I-mutant
2	I-mutant
,	O
which	O
deviates	O
from	O
L1	B-mutant
-	I-mutant
12	I-mutant
-	I-mutant
1	I-mutant
at	O
residues	O
29	O
-	O
32	O
,	O
i	O
.	O
e	O
.,	O
at	O
the	O
site	O
of	O
insertion	O
with	O
respect	O
to	O
the	O
11	O
-	O
residue	O
CDR	O
.	O
	O
The	O
fourth	O
member	O
of	O
the	O
set	O
,	O
H1	O
-	O
69	O
:	O
L3	O
-	O
20	O
,	O
was	O
crystallized	O
with	O
2	O
Fabs	O
in	O
the	O
asymmetric	O
unit	O
.	O
	O
As	O
mentioned	O
earlier	O
,	O
all	O
16	O
Fabs	O
have	O
the	O
same	O
CDR	O
H3	O
,	O
for	O
which	O
the	O
amino	O
acid	O
sequence	O
is	O
derived	O
from	O
the	O
anti	O
-	O
CCL2	O
antibody	O
CNTO	O
888	O
.	O
	O
The	O
variations	O
in	O
CDR	O
H3	O
conformation	O
are	O
illustrated	O
in	O
Fig	O
.	O
6	O
for	O
the	O
18	O
Fab	O
structures	O
that	O
have	O
ordered	O
backbone	O
atoms	O
.	O
	O
(	O
B	O
)	O
The	O
“	O
extended	O
”	O
CDR	O
H3	O
of	O
H1	O
-	O
69	O
:	O
L3	O
-	O
20	O
with	O
green	O
carbon	O
atoms	O
and	O
yellow	O
dashed	O
lines	O
connecting	O
the	O
H	O
-	O
bond	O
pairs	O
for	O
Asp101	O
OD1	O
and	O
OD2	O
and	O
Trp103	O
NE1	O
.	O
	O
The	O
remaining	O
8	O
Fabs	O
can	O
be	O
grouped	O
into	O
5	O
different	O
conformational	O
classes	O
.	O
	O
Position	O
43	O
may	O
be	O
alternatively	O
occupied	O
by	O
Ser	O
,	O
Val	O
or	O
Pro	O
(	O
as	O
in	O
L4	B-mutant
-	I-mutant
1	I-mutant
),	O
but	O
the	O
hydrophobic	O
interaction	O
with	O
H	O
-	O
Tyr91	O
is	O
preserved	O
.	O
	O
In	O
most	O
of	O
the	O
structures	O
,	O
it	O
has	O
the	O
χ2	O
angle	O
of	O
∼	O
80	O
°,	O
while	O
the	O
ring	O
is	O
flipped	O
over	O
(	O
χ2	O
=	O
−	O
100	O
°)	O
in	O
H5	O
-	O
51	O
:	O
L3	O
:	O
11	O
and	O
H5	O
-	O
51	O
:	O
L3	O
-	O
20	O
.	O
	O
In	O
fact	O
,	O
the	O
parameter	O
values	O
for	O
the	O
set	O
of	O
16	O
Fabs	O
are	O
in	O
the	O
middle	O
of	O
the	O
distribution	O
observed	O
for	O
351	O
non	O
-	O
redundant	O
antibody	O
structures	O
determined	O
at	O
3	O
.	O
0	O
Å	O
resolution	O
or	O
better	O
.	O
	O
An	O
illustration	O
of	O
the	O
difference	O
in	O
tilt	O
angle	O
for	O
2	O
pairs	O
of	O
variants	O
by	O
the	O
superposition	O
of	O
the	O
VH	O
domains	O
of	O
(	O
A	O
)	O
H1	O
-	O
69	O
:	O
L3	O
-	O
20	O
on	O
that	O
of	O
H5	O
-	O
51	O
:	O
L1	O
-	O
39	O
(	O
the	O
VL	O
domain	O
is	O
off	O
by	O
a	O
rigid	O
-	O
body	O
roatation	O
of	O
10	O
.	O
5	O
°)	O
and	O
(	O
B	O
)	O
H1	O
-	O
69	O
:	O
L4	O
-	O
1	O
on	O
that	O
of	O
H5	O
-	O
51	O
:	O
L1	O
-	O
39	O
(	O
the	O
VL	O
domain	O
is	O
off	O
by	O
a	O
rigid	O
-	O
body	O
roatation	O
of	O
1	O
.	O
6	O
°).	O
	O
One	O
of	O
the	O
2	O
structures	O
,	O
H1	O
-	O
69	O
:	O
L3	O
-	O
20	O
,	O
has	O
its	O
CDR	O
H3	O
in	O
the	O
‘	O
extended	O
’	O
conformation	O
;	O
the	O
other	O
structure	O
has	O
it	O
in	O
the	O
‘	O
kinked	O
’	O
conformation	O
.	O
	O
VH	O
:	O
VL	O
buried	O
surface	O
area	O
and	O
complementarity	O
	O
Residues	O
in	O
CDR	O
H3	O
are	O
missing	O
:	O
YGE	O
in	O
H5	O
-	O
51	O
:	O
L3	O
-	O
11	O
,	O
GIY	O
in	O
H5	O
-	O
51	O
:	O
L3	O
-	O
20	O
.	O
	O
This	O
is	O
the	O
first	O
report	O
of	O
a	O
systematic	O
structural	O
investigation	O
of	O
a	O
phage	O
germline	O
library	O
.	O
	O
The	O
16	O
Fab	O
structures	O
offer	O
a	O
unique	O
look	O
at	O
all	O
pairings	O
of	O
4	O
different	O
HCs	O
(	O
H1	B-mutant
-	I-mutant
69	I-mutant
,	O
H3	B-mutant
-	I-mutant
23	I-mutant
,	O
H3	B-mutant
-	I-mutant
53	I-mutant
,	O
and	O
H5	B-mutant
-	I-mutant
51	I-mutant
)	O
and	O
4	O
different	O
LCs	O
(	O
L1	B-mutant
-	I-mutant
39	I-mutant
,	O
L3	B-mutant
-	I-mutant
11	I-mutant
,	O
L3	B-mutant
-	I-mutant
20	I-mutant
and	O
L4	B-mutant
-	I-mutant
1	I-mutant
),	O
all	O
with	O
the	O
same	O
CDR	O
H3	O
.	O
	O
Having	O
all	O
16	O
VH	O
:	O
VL	O
pairs	O
with	O
the	O
same	O
CDR	O
H3	O
provides	O
some	O
insights	O
into	O
why	O
molecular	O
modeling	O
efforts	O
of	O
CDR	O
H3	O
have	O
proven	O
so	O
difficult	O
.	O
	O
Thus	O
,	O
it	O
is	O
likely	O
that	O
the	O
CDR	O
H3	O
conformation	O
is	O
dependent	O
upon	O
2	O
dominating	O
factors	O
:	O
1	O
)	O
amino	O
acid	O
sequence	O
;	O
and	O
2	O
)	O
VH	O
and	O
VL	O
context	O
.	O
	O
This	O
subset	O
also	O
has	O
2	O
structures	O
with	O
2	O
Fab	O
copies	O
in	O
the	O
asymmetric	O
unit	O
.	O
	O
The	O
same	O
variability	O
is	O
observed	O
for	O
the	O
sets	O
of	O
variants	O
composed	O
of	O
one	O
LC	O
paired	O
with	O
each	O
of	O
the	O
4	O
HCs	O
.	O
	O
As	O
noted	O
in	O
the	O
Results	O
section	O
,	O
the	O
2	O
variants	O
,	O
H1	O
-	O
69	O
:	O
L3	O
-	O
20	O
and	O
H3	O
-	O
23	O
:	O
L3	O
-	O
20	O
,	O
are	O
outliers	O
in	O
terms	O
of	O
the	O
tilt	O
angle	O
;	O
at	O
the	O
same	O
time	O
,	O
both	O
have	O
the	O
smallest	O
VH	O
:	O
VL	O
interface	O
.	O
	O
Other	O
germlines	O
have	O
bulky	O
residues	O
,	O
Tyr	O
,	O
Arg	O
and	O
Trp	O
,	O
at	O
these	O
positions	O
,	O
whereas	O
L1	B-mutant
-	I-mutant
39	I-mutant
has	O
Ser	O
and	O
Thr	O
.	O
	O
A	O
more	O
compact	O
CDR	O
L3	O
may	O
be	O
beneficial	O
in	O
this	O
situation	O
.	O
	O
Yet	O
,	O
for	O
the	O
2	O
antibodies	O
,	O
the	O
total	O
gain	O
in	O
stability	O
merits	O
the	O
domain	O
repacking	O
.	O
	O
Quite	O
unexpectedly	O
,	O
2	O
of	O
the	O
variants	O
,	O
H1	O
-	O
69	O
:	O
L3	O
-	O
20	O
and	O
H3	O
-	O
53	O
:	O
L4	O
-	O
1	O
,	O
have	O
the	O
‘	O
extended	O
’	O
stem	O
region	O
differing	O
from	O
the	O
other	O
14	O
that	O
have	O
a	O
‘	O
kinked	O
’	O
stem	O
region	O
.	O
	O
From	O
this	O
point	O
of	O
view	O
,	O
a	O
novel	O
approach	O
to	O
design	O
combinatorial	O
antibody	O
libraries	O
would	O
be	O
to	O
cover	O
the	O
range	O
of	O
CDR	O
conformations	O
that	O
may	O
not	O
necessarily	O
coincide	O
with	O
the	O
germline	O
usage	O
in	O
the	O
human	O
repertoire	O
.	O
	O
This	O
study	O
resulted	O
in	O
a	O
series	O
of	O
snapshots	O
depicting	O
the	O
various	O
folding	O
states	O
of	O
Im7	O
while	O
bound	O
to	O
Spy	O
.	O
	O
Recent	O
advances	O
in	O
X	O
-	O
ray	O
crystallography	O
and	O
NMR	O
spectroscopy	O
continue	O
to	O
improve	O
our	O
ability	O
to	O
analyze	O
biomolecules	O
that	O
exist	O
in	O
multiple	O
conformations	O
.	O
	O
X	O
-	O
ray	O
crystallography	O
has	O
historically	O
provided	O
valuable	O
information	O
on	O
small	O
-	O
scale	O
conformational	O
changes	O
,	O
but	O
observing	O
large	O
-	O
amplitude	O
heterogeneous	O
conformational	O
changes	O
often	O
falls	O
beyond	O
the	O
reach	O
of	O
current	O
crystallographic	O
techniques	O
.	O
	O
However	O
,	O
modeling	O
of	O
the	O
substrate	O
in	O
the	O
complex	O
proved	O
to	O
be	O
a	O
substantial	O
challenge	O
,	O
as	O
the	O
electron	O
density	O
of	O
the	O
substrate	O
was	O
discontinuous	O
and	O
fragmented	O
.	O
	O
To	O
determine	O
the	O
structure	O
of	O
the	O
substrate	O
portion	O
of	O
these	O
Spy	O
:	O
substrate	O
complexes	O
,	O
we	O
conceived	O
of	O
an	O
approach	O
that	O
we	O
term	O
READ	O
,	O
for	O
Residual	O
Electron	O
and	O
Anomalous	O
Density	O
.	O
	O
Its	O
strong	O
anomalous	O
scattering	O
allowed	O
us	O
to	O
track	O
the	O
positions	O
of	O
these	O
individual	O
Im76	B-mutant
-	I-mutant
45	I-mutant
residues	O
one	O
at	O
a	O
time	O
,	O
potentially	O
even	O
if	O
the	O
residue	O
was	O
found	O
in	O
several	O
locations	O
in	O
the	O
same	O
crystal	O
.	O
	O
Together	O
,	O
these	O
results	O
indicated	O
that	O
the	O
Im7	O
substrate	O
binds	O
Spy	O
in	O
multiple	O
conformations	O
.	O
	O
To	O
generate	O
an	O
accurate	O
depiction	O
of	O
the	O
chaperone	O
-	O
substrate	O
interactions	O
,	O
we	O
devised	O
a	O
selection	O
protocol	O
based	O
on	O
a	O
sample	O
-	O
and	O
-	O
select	O
procedure	O
employed	O
in	O
NMR	O
spectroscopy	O
.	O
	O
The	O
coarse	O
-	O
grained	O
simulations	O
are	O
based	O
on	O
a	O
single	O
-	O
residue	O
resolution	O
model	O
for	O
protein	O
folding	O
and	O
were	O
extended	O
here	O
to	O
describe	O
Spy	O
-	O
Im76	O
-	O
45	O
binding	O
events	O
(	O
Online	O
Methods	O
).	O
	O
To	O
accomplish	O
this	O
task	O
,	O
we	O
generated	O
a	O
compressed	O
version	O
of	O
the	O
experimental	O
2mFo	O
−	O
DFc	O
electron	O
density	O
map	O
for	O
use	O
in	O
the	O
selection	O
.	O
	O
We	O
constructed	O
a	O
contact	O
map	O
of	O
the	O
complex	O
,	O
which	O
shows	O
the	O
frequency	O
of	O
interactions	O
for	O
chaperone	O
-	O
substrate	O
residue	O
pairs	O
(	O
Fig	O
.	O
4	O
).	O
	O
The	O
Spy	O
-	O
contacting	O
residues	O
comprise	O
a	O
mixture	O
of	O
charged	O
,	O
polar	O
,	O
and	O
hydrophobic	O
residues	O
.	O
	O
Once	O
the	O
substrate	O
begins	O
to	O
fold	O
within	O
this	O
protected	O
environment	O
,	O
it	O
progressively	O
buries	O
its	O
own	O
hydrophobic	O
residues	O
,	O
and	O
its	O
interactions	O
with	O
the	O
chaperone	O
shift	O
towards	O
becoming	O
more	O
electrostatic	O
.	O
	O
Residues	O
Asp32	O
and	O
Asp35	O
are	O
close	O
to	O
each	O
other	O
in	O
the	O
folded	O
state	O
of	O
Im7	O
.	O
	O
This	O
proximity	O
likely	O
causes	O
electrostatic	O
repulsion	O
that	O
destabilizes	O
Im7	O
’	O
s	O
native	O
state	O
.	O
	O
In	O
conjunction	O
with	O
our	O
bound	O
Im76	B-mutant
-	I-mutant
45	I-mutant
ensemble	O
,	O
these	O
mutants	O
now	O
allowed	O
us	O
to	O
investigate	O
structural	O
features	O
important	O
to	O
chaperone	O
function	O
.	O
	O
Despite	O
extensive	O
studies	O
,	O
exactly	O
how	O
complex	O
chaperone	O
machines	O
help	O
proteins	O
fold	O
remains	O
controversial	O
.	O
	O
Heterogeneous	O
dynamic	O
complexes	O
or	O
disordered	O
regions	O
of	O
single	O
proteins	O
,	O
once	O
considered	O
solely	O
approachable	O
by	O
NMR	O
spectroscopy	O
,	O
can	O
now	O
be	O
visualized	O
through	O
X	O
-	O
ray	O
crystallography	O
.	O
	O
Flowchart	O
of	O
the	O
READ	O
sample	O
-	O
and	O
-	O
select	O
process	O
.	O
	O
(	O
a	O
)	O
Spy	O
:	O
Im76	O
-	O
45	O
contact	O
map	O
projected	O
onto	O
the	O
bound	O
Spy	O
dimer	O
(	O
above	O
)	O
and	O
Im76	B-mutant
-	I-mutant
45	I-mutant
(	O
below	O
)	O
structures	O
.	O
	O
(	O
a	O
)	O
Overlay	O
of	O
apo	O
Spy	O
(	O
PDB	O
ID	O
:	O
3O39	O
,	O
gray	O
)	O
and	O
bound	O
Spy	O
(	O
green	O
).	O
(	O
b	O
)	O
Overlay	O
of	O
WT	O
Spy	O
bound	O
to	O
Im76	B-mutant
-	I-mutant
45	I-mutant
(	O
green	O
),	O
H96L	B-mutant
Spy	O
bound	O
to	O
Im7	O
L18A	B-mutant
L19	B-mutant
AL13A	I-mutant
(	O
blue	O
),	O
H96L	B-mutant
Spy	O
bound	O
to	O
WT	O
Im7	O
(	O
yellow	O
),	O
and	O
WT	O
Spy	O
bound	O
to	O
casein	O
(	O
salmon	O
).	O
(	O
c	O
)	O
Competition	O
assay	O
showing	O
Im76	B-mutant
-	I-mutant
45	I-mutant
competes	O
with	O
Im7	O
L18A	B-mutant
L19A	B-mutant
L37A	B-mutant
H40W	B-mutant
for	O
the	O
same	O
binding	O
site	O
on	O
Spy	O
(	O
further	O
substrate	O
competition	O
assays	O
are	O
shown	O
in	O
Supplementary	O
Fig	O
.	O
8	O
).	O
	O
(	O
b	O
)	O
F115	O
and	O
L32	O
tether	O
Spy	O
’	O
s	O
linker	O
region	O
to	O
its	O
cradle	O
,	O
decreasing	O
Spy	O
activity	O
by	O
limiting	O
linker	O
region	O
flexibility	O
.	O
	O
Despite	O
a	O
long	O
history	O
of	O
physiological	O
and	O
functional	O
studies	O
,	O
the	O
molecular	O
mechanism	O
of	O
NCX	O
has	O
been	O
elusive	O
,	O
owing	O
to	O
the	O
lack	O
of	O
structural	O
information	O
.	O
	O
In	O
this	O
study	O
,	O
we	O
set	O
out	O
to	O
determine	O
the	O
structures	O
of	O
outward	O
-	O
facing	O
wild	O
-	O
type	O
NCX_Mj	O
in	O
complex	O
with	O
Na	O
+,	O
Ca2	O
+	O
and	O
Sr2	O
+,	O
at	O
various	O
concentrations	O
.	O
	O
Extracellular	O
Na	O
+	O
binding	O
	O
To	O
conclusively	O
clarify	O
this	O
assignment	O
,	O
we	O
first	O
set	O
out	O
to	O
examine	O
the	O
Na	O
+	O
occupancy	O
of	O
these	O
sites	O
without	O
Ca2	O
+.	O
	O
X	O
-	O
ray	O
diffraction	O
of	O
these	O
soaked	O
crystals	O
revealed	O
a	O
Na	O
+-	O
dependent	O
variation	O
in	O
the	O
electron	O
-	O
density	O
distribution	O
at	O
sites	O
Sext	O
,	O
SCa	O
and	O
Sint	O
,	O
indicating	O
a	O
Na	O
+	O
occupancy	O
change	O
(	O
Fig	O
.	O
1c	O
).	O
	O
Indeed	O
,	O
two	O
observations	O
indicate	O
that	O
a	O
water	O
molecule	O
rather	O
than	O
a	O
Na	O
+	O
ion	O
occupies	O
Smid	O
,	O
as	O
was	O
predicted	O
in	O
a	O
recent	O
simulation	O
study	O
.	O
	O
When	O
Na	O
+	O
binds	O
to	O
Sext	O
at	O
high	O
concentrations	O
,	O
the	O
N	O
-	O
terminal	O
half	O
of	O
TM7	O
is	O
bent	O
into	O
two	O
short	O
helices	O
,	O
TM7a	O
and	O
TM7b	O
(	O
Fig	O
.	O
2a	O
).	O
	O
TM7b	O
occludes	O
the	O
four	O
central	O
binding	O
sites	O
from	O
the	O
external	O
solution	O
,	O
with	O
the	O
backbone	O
carbonyl	O
of	O
Ala206	O
coordinating	O
the	O
Na	O
+	O
ion	O
(	O
Fig	O
.	O
2b	O
-	O
d	O
).	O
	O
Extracellular	O
Ca2	O
+	O
and	O
Sr2	O
+	O
binding	O
and	O
their	O
competition	O
with	O
Na	O
+	O
	O
Binding	O
of	O
Ca2	O
+	O
to	O
both	O
sites	O
simultaneously	O
is	O
highly	O
improbable	O
due	O
to	O
their	O
close	O
proximity	O
,	O
and	O
at	O
least	O
one	O
water	O
molecule	O
can	O
be	O
discerned	O
coordinating	O
the	O
ion	O
(	O
Fig	O
.	O
3b	O
).	O
	O
Indeed	O
,	O
in	O
most	O
NCX	O
proteins	O
Asp240	O
is	O
substituted	O
by	O
Asn	O
,	O
which	O
would	O
likely	O
weaken	O
or	O
abrogate	O
Ca2	O
+	O
binding	O
to	O
Smid	O
.	O
	O
Although	O
the	O
binding	O
sites	O
are	O
thus	O
fully	O
accessible	O
to	O
the	O
external	O
solution	O
(	O
Fig	O
.	O
3e	O
),	O
the	O
lack	O
of	O
electron	O
density	O
therein	O
indicates	O
no	O
ions	O
or	O
ordered	O
solvent	O
molecules	O
.	O
	O
Such	O
interpretation	O
would	O
be	O
consistent	O
with	O
the	O
computer	O
simulations	O
reported	O
below	O
.	O
	O
That	O
secondary	O
-	O
active	O
transporters	O
are	O
able	O
to	O
harness	O
an	O
electrochemical	O
gradient	O
of	O
one	O
substrate	O
to	O
power	O
the	O
uphill	O
transport	O
of	O
another	O
relies	O
on	O
a	O
seemingly	O
simple	O
principle	O
:	O
they	O
must	O
not	O
transition	O
between	O
outward	O
-	O
and	O
inward	O
-	O
open	O
conformations	O
unless	O
in	O
two	O
precise	O
substrate	O
occupancy	O
states	O
.	O
	O
As	O
it	O
happens	O
,	O
the	O
results	O
confirm	O
that	O
the	O
structures	O
now	O
available	O
are	O
representing	O
interconverting	O
states	O
of	O
the	O
functional	O
cycle	O
of	O
NCX_Mj	O
,	O
while	O
revealing	O
how	O
the	O
alternating	O
-	O
access	O
mechanism	O
is	O
controlled	O
by	O
the	O
ion	O
-	O
occupancy	O
state	O
.	O
	O
This	O
distortion	O
occludes	O
Sext	O
from	O
the	O
exterior	O
(	O
Fig	O
.	O
4d	O
,	O
4h	O
-	O
i	O
)	O
and	O
appears	O
to	O
be	O
induced	O
by	O
the	O
Na	O
+	O
ion	O
itself	O
,	O
which	O
pulls	O
the	O
carbonyl	O
group	O
of	O
A206	O
into	O
its	O
coordination	O
sphere	O
(	O
Fig	O
.	O
4g	O
).	O
	O
When	O
all	O
Na	O
+	O
sites	O
are	O
occupied	O
,	O
the	O
global	O
free	O
-	O
energy	O
minimum	O
corresponds	O
to	O
a	O
conformation	O
in	O
which	O
the	O
ions	O
are	O
maximally	O
coordinated	O
by	O
the	O
protein	O
(	O
Fig	O
.	O
5a	O
,	O
5c	O
);	O
TM7ab	O
is	O
bent	O
and	O
packs	O
closely	O
with	O
TM2	O
and	O
TM3	O
,	O
and	O
so	O
the	O
binding	O
sites	O
are	O
occluded	O
from	O
the	O
solvent	O
(	O
Fig	O
.	O
5b	O
).	O
	O
The	O
Na	O
+	O
ion	O
at	O
Sext	O
remains	O
fully	O
coordinated	O
,	O
but	O
an	O
ordered	O
water	O
molecule	O
now	O
mediates	O
its	O
interaction	O
with	O
A206	O
:	O
O	O
,	O
relieving	O
the	O
strain	O
on	O
the	O
F202	O
:	O
O	O
–	O
A206	O
:	O
N	O
hydrogen	O
-	O
bond	O
(	O
Fig	O
.	O
5c	O
).	O
	O
Interestingly	O
,	O
this	O
doubly	O
occupied	O
state	O
can	O
also	O
access	O
conformations	O
in	O
which	O
the	O
second	O
aqueous	O
channel	O
mentioned	O
above	O
,	O
i	O
.	O
e	O
.	O
leading	O
to	O
SCa	O
between	O
TM7	O
and	O
TM2	O
and	O
over	O
the	O
gating	O
helices	O
TM1	O
and	O
TM6	O
,	O
also	O
becomes	O
open	O
(	O
Fig	O
.	O
5b	O
-	O
c	O
).	O
	O
This	O
processivity	O
is	O
logical	O
since	O
three	O
Na	O
+	O
ions	O
are	O
involved	O
,	O
but	O
also	O
implies	O
that	O
in	O
the	O
Ca2	O
+-	O
bound	O
state	O
,	O
which	O
includes	O
a	O
single	O
ion	O
,	O
the	O
transporter	O
ought	O
to	O
be	O
able	O
to	O
access	O
all	O
three	O
major	O
conformations	O
,	O
i	O
.	O
e	O
.	O
the	O
outward	O
-	O
open	O
state	O
,	O
in	O
order	O
to	O
release	O
(	O
or	O
re	O
-	O
bind	O
)	O
Ca2	O
+,	O
but	O
also	O
the	O
occluded	O
conformation	O
,	O
and	O
thus	O
the	O
semi	O
-	O
open	O
intermediate	O
,	O
in	O
order	O
to	O
transition	O
to	O
the	O
inward	O
-	O
open	O
state	O
.	O
	O
By	O
contrast	O
,	O
occupancy	O
by	O
H	O
+,	O
which	O
as	O
mentioned	O
are	O
not	O
transported	O
,	O
might	O
be	O
compatible	O
with	O
a	O
semi	O
-	O
open	O
state	O
as	O
well	O
as	O
with	O
the	O
fully	O
open	O
conformation	O
,	O
but	O
should	O
not	O
be	O
conducive	O
to	O
occlusion	O
.	O
	O
This	O
occluded	O
conformation	O
,	O
which	O
is	O
a	O
necessary	O
intermediate	O
between	O
the	O
outward	O
and	O
inward	O
-	O
open	O
states	O
,	O
and	O
which	O
entails	O
the	O
internal	O
dehydration	O
of	O
the	O
protein	O
,	O
is	O
only	O
attainable	O
upon	O
complete	O
occupancy	O
of	O
the	O
binding	O
sites	O
.	O
	O
The	O
most	O
apparent	O
of	O
these	O
changes	O
involves	O
the	O
N	O
-	O
terminal	O
half	O
of	O
TM7	O
(	O
TM7ab	O
);	O
together	O
with	O
more	O
subtle	O
displacements	O
in	O
TM2	O
and	O
TM3	O
,	O
this	O
change	O
in	O
TM7ab	O
correlates	O
with	O
the	O
opening	O
and	O
closing	O
of	O
two	O
distinct	O
aqueous	O
channels	O
leading	O
into	O
the	O
ion	O
-	O
binding	O
sites	O
from	O
the	O
extracellular	O
solution	O
.	O
	O
The	O
striking	O
quantitative	O
agreement	O
between	O
the	O
ion	O
-	O
binding	O
affinities	O
inferred	O
from	O
our	O
crystallographic	O
titrations	O
and	O
the	O
Km	O
and	O
K1	O
/	O
2	O
values	O
previously	O
deduced	O
from	O
functional	O
assays	O
has	O
been	O
discussed	O
above	O
.	O
	O
Specifically	O
,	O
our	O
crystal	O
titrations	O
suggest	O
that	O
,	O
during	O
forward	O
Na	O
+/	O
Ca2	O
+	O
exchange	O
,	O
sites	O
Sint	O
and	O
SCa	O
,	O
which	O
Ca2	O
+	O
and	O
Na	O
+	O
compete	O
for	O
,	O
can	O
be	O
grouped	O
into	O
one	O
;	O
Na	O
+	O
binding	O
to	O
these	O
sites	O
does	O
not	O
require	O
high	O
Na	O
+	O
concentrations	O
,	O
and	O
two	O
Na	O
+	O
ions	O
along	O
with	O
a	O
water	O
molecule	O
(	O
at	O
Smid	O
)	O
are	O
sufficient	O
to	O
displace	O
Ca2	O
+,	O
explaining	O
the	O
Hill	O
coefficient	O
of	O
~	O
2	O
for	O
Na	O
+-	O
dependent	O
inhibition	O
of	O
Ca2	O
+	O
fluxes	O
.	O
	O
No	O
significant	O
changes	O
were	O
observed	O
in	O
the	O
side	O
-	O
chains	O
involved	O
in	O
ion	O
or	O
water	O
coordination	O
at	O
the	O
SCa	O
,	O
Sint	O
and	O
Smid	O
sites	O
.	O
	O
The	O
vacant	O
Sext	O
site	O
in	O
the	O
structure	O
at	O
low	O
Na	O
+	O
concentration	O
is	O
indicated	O
with	O
a	O
white	O
sphere	O
.	O
	O
(	O
d	O
)	O
Extracellular	O
solvent	O
accessibility	O
of	O
the	O
ion	O
binding	O
sites	O
in	O
the	O
structures	O
at	O
high	O
and	O
low	O
[	O
Na	O
+].	O
	O
Putative	O
solvent	O
channels	O
are	O
represented	O
as	O
light	O
-	O
purple	O
surfaces	O
.	O
	O
Residues	O
involved	O
in	O
Sr2	O
+	O
coordination	O
are	O
labeled	O
.	O
	O
(	O
b	O
)	O
Ca2	O
+	O
(	O
tanned	O
spheres	O
)	O
binds	O
either	O
to	O
SCa	O
or	O
Smid	O
in	O
crystals	O
titrated	O
with	O
10	O
mM	O
Ca2	O
+	O
and	O
2	O
.	O
5	O
mM	O
Na	O
+	O
(	O
see	O
also	O
Supplementary	O
Fig	O
.	O
2	O
).	O
	O
Approximate	O
distances	O
between	O
TM2	O
,	O
TM3	O
and	O
TM7	O
are	O
indicated	O
in	O
Å	O
.	O
(	O
e	O
)	O
Close	O
-	O
up	O
of	O
the	O
ion	O
-	O
binding	O
region	O
in	O
the	O
partially	O
Na	O
+-	O
occupied	O
state	O
.	O
	O
The	O
water	O
-	O
density	O
maps	O
in	O
(	O
b	O
)	O
are	O
shown	O
here	O
as	O
a	O
grey	O
mesh	O
.	O
	O
An	O
extended	O
U2AF65	O
–	O
RNA	O
-	O
binding	O
domain	O
recognizes	O
the	O
3	O
′	O
splice	O
site	O
signal	O
	O
Initially	O
U2AF65	O
recognizes	O
the	O
Py	O
-	O
tract	O
splice	O
site	O
signal	O
.	O
	O
As	O
such	O
,	O
the	O
molecular	O
mechanisms	O
for	O
Py	O
-	O
tract	O
recognition	O
by	O
the	O
intact	O
U2AF65	O
–	O
RNA	O
-	O
binding	O
domain	O
remained	O
unknown	O
.	O
	O
We	O
use	O
single	O
-	O
molecule	O
Förster	O
resonance	O
energy	O
transfer	O
(	O
smFRET	O
)	O
to	O
characterize	O
the	O
conformational	O
dynamics	O
of	O
this	O
extended	O
U2AF65	O
–	O
RNA	O
-	O
binding	O
domain	O
during	O
Py	O
-	O
tract	O
recognition	O
.	O
	O
The	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
RRM1	O
and	O
RRM2	O
associate	O
with	O
the	O
Py	O
tract	O
in	O
a	O
parallel	O
,	O
side	O
-	O
by	O
-	O
side	O
arrangement	O
(	O
shown	O
for	O
representative	O
structure	O
iv	O
in	O
Fig	O
.	O
2b	O
,	O
c	O
;	O
Supplementary	O
Movie	O
1	O
).	O
	O
An	O
extended	O
conformation	O
of	O
the	O
U2AF65	O
inter	O
-	O
RRM	O
linker	O
traverses	O
across	O
the	O
α	O
-	O
helical	O
surface	O
of	O
RRM1	O
and	O
the	O
central	O
β	O
-	O
strands	O
of	O
RRM2	O
and	O
is	O
well	O
defined	O
in	O
the	O
electron	O
density	O
(	O
Fig	O
.	O
2b	O
).	O
	O
Both	O
RRM1	O
/	O
RRM2	O
extensions	O
and	O
the	O
inter	O
-	O
RRM	O
linker	O
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
directly	O
recognize	O
the	O
bound	O
oligonucleotide	O
.	O
	O
Based	O
on	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
structures	O
,	O
we	O
originally	O
hypothesized	O
that	O
the	O
U2AF65	O
RRMs	O
would	O
bind	O
the	O
minimal	O
seven	O
nucleotides	O
observed	O
in	O
these	O
structures	O
.	O
	O
Surprisingly	O
,	O
the	O
RRM2	O
extension	O
/	O
inter	O
-	O
RRM	O
linker	O
contribute	O
new	O
central	O
nucleotide	O
-	O
binding	O
sites	O
near	O
the	O
RRM1	O
/	O
RRM2	O
junction	O
and	O
the	O
RRM1	O
extension	O
recognizes	O
the	O
3	O
′-	O
terminal	O
nucleotide	O
(	O
Fig	O
.	O
2c	O
;	O
Supplementary	O
Movie	O
1	O
).	O
	O
Qualitatively	O
,	O
a	O
subset	O
of	O
the	O
U2AF651	O
,	O
2L	O
-	O
nucleotide	O
-	O
binding	O
sites	O
(	O
sites	O
1	O
–	O
3	O
and	O
7	O
–	O
9	O
)	O
share	O
similar	O
locations	O
to	O
those	O
of	O
the	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
structures	O
(	O
Supplementary	O
Figs	O
2c	O
,	O
d	O
and	O
3	O
).	O
	O
Otherwise	O
,	O
the	O
rU4	O
nucleotide	O
packs	O
against	O
F304	O
in	O
the	O
signature	O
ribonucleoprotein	O
consensus	O
motif	O
(	O
RNP	O
)-	O
2	O
of	O
RRM2	O
.	O
	O
This	O
nucleotide	O
twists	O
to	O
face	O
away	O
from	O
the	O
U2AF65	O
linker	O
and	O
instead	O
inserts	O
the	O
rU6	O
-	O
uracil	O
into	O
a	O
sandwich	O
between	O
the	O
β2	O
/	O
β3	O
loops	O
of	O
RRM1	O
and	O
RRM2	O
.	O
	O
The	O
N	O
-	O
and	O
C	O
-	O
terminal	O
extensions	O
of	O
the	O
U2AF65	O
RRM1	O
and	O
RRM2	O
directly	O
contact	O
the	O
bound	O
Py	O
tract	O
.	O
	O
Consequently	O
,	O
the	O
U2AF651	O
,	O
2L	O
-	O
bound	O
rU2	O
-	O
O4	O
and	O
-	O
N3H	O
form	O
dual	O
hydrogen	O
bonds	O
with	O
the	O
K329	O
backbone	O
atoms	O
(	O
Fig	O
.	O
3a	O
),	O
rather	O
than	O
a	O
single	O
hydrogen	O
bond	O
with	O
the	O
K329	O
side	O
chain	O
as	O
in	O
the	O
prior	O
dU2AF651	B-mutant
,	I-mutant
2	I-mutant
structure	O
(	O
Supplementary	O
Fig	O
.	O
3b	O
).	O
	O
The	O
adjacent	O
R146	O
guanidinium	O
group	O
donates	O
hydrogen	O
bonds	O
to	O
the	O
3	O
′-	O
terminal	O
ribose	O
-	O
O2	O
′	O
and	O
O3	O
′	O
atoms	O
,	O
where	O
it	O
could	O
form	O
a	O
salt	O
bridge	O
with	O
a	O
phospho	O
-	O
diester	O
group	O
in	O
the	O
context	O
of	O
a	O
longer	O
pre	O
-	O
mRNA	O
.	O
	O
We	O
compare	O
U2AF65	O
interactions	O
with	O
uracil	O
relative	O
to	O
cytosine	O
pyrimidines	O
at	O
the	O
ninth	O
binding	O
site	O
in	O
Fig	O
.	O
3g	O
,	O
h	O
and	O
the	O
Supplementary	O
Discussion	O
.	O
	O
At	O
the	O
RNA	O
surface	O
,	O
the	O
key	O
V254	O
that	O
recognizes	O
the	O
fifth	O
uracil	O
is	O
secured	O
via	O
hydrophobic	O
contacts	O
between	O
its	O
side	O
chain	O
and	O
the	O
β	O
-	O
sheet	O
surface	O
of	O
RRM2	O
,	O
chiefly	O
the	O
consensus	O
RNP1	O
-	O
F304	O
residue	O
that	O
stacks	O
with	O
the	O
fourth	O
uracil	O
(	O
Fig	O
.	O
4a	O
,	O
lower	O
left	O
).	O
	O
However	O
,	O
the	O
resulting	O
decrease	O
in	O
the	O
AdML	O
RNA	O
affinity	O
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
-	I-mutant
3Gly	I-mutant
mutant	O
relative	O
to	O
wild	O
-	O
type	O
protein	O
was	O
not	O
significant	O
(	O
Fig	O
.	O
4b	O
).	O
	O
In	O
parallel	O
,	O
we	O
replaced	O
five	O
linker	O
residues	O
(	O
S251	O
,	O
T252	O
,	O
V253	O
,	O
V254	O
and	O
P255	O
)	O
at	O
the	O
fifth	O
nucleotide	O
-	O
binding	O
site	O
with	O
glycines	O
(	O
5Gly	B-mutant
)	O
and	O
also	O
found	O
that	O
the	O
RNA	O
affinity	O
of	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
-	I-mutant
5Gly	I-mutant
mutant	O
likewise	O
decreased	O
only	O
slightly	O
relative	O
to	O
wild	O
-	O
type	O
protein	O
.	O
	O
Importance	O
of	O
U2AF65	O
–	O
RNA	O
contacts	O
for	O
pre	O
-	O
mRNA	O
splicing	O
	O
To	O
complement	O
the	O
static	O
portraits	O
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structure	O
that	O
we	O
had	O
determined	O
by	O
X	O
-	O
ray	O
crystallography	O
,	O
we	O
used	O
smFRET	O
to	O
characterize	O
the	O
probability	O
distribution	O
functions	O
and	O
time	O
dependence	O
of	O
U2AF65	O
inter	O
-	O
RRM	O
conformational	O
dynamics	O
in	O
solution	O
.	O
	O
Double	O
-	O
cysteine	O
variant	O
of	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
was	O
modified	O
with	O
equimolar	O
amount	O
of	O
Cy3	O
and	O
Cy5	O
.	O
	O
Only	O
traces	O
that	O
showed	O
single	O
photobleaching	O
events	O
for	O
both	O
donor	O
and	O
acceptor	O
dyes	O
and	O
anti	O
-	O
correlated	O
changes	O
in	O
acceptor	O
and	O
donor	O
fluorescence	O
were	O
included	O
in	O
smFRET	O
data	O
analysis	O
.	O
	O
The	O
double	O
-	O
labelled	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	O
/	O
Cy5	O
)	O
protein	O
was	O
tethered	O
to	O
a	O
slide	O
via	O
biotin	O
-	O
NTA	O
/	O
Ni	O
+	O
2	O
resin	O
.	O
	O
However	O
,	O
the	O
presence	O
of	O
repetitive	O
fluctuations	O
between	O
particular	O
FRET	O
values	O
supports	O
the	O
hypothesis	O
that	O
RNA	O
-	O
free	O
U2AF65	O
samples	O
several	O
distinct	O
conformations	O
.	O
	O
This	O
result	O
is	O
consistent	O
with	O
the	O
broad	O
ensembles	O
of	O
extended	O
solution	O
conformations	O
that	O
best	O
fit	O
the	O
SAXS	O
data	O
collected	O
for	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
as	O
well	O
as	O
for	O
a	O
longer	O
construct	O
(	O
residues	O
136	O
–	O
347	O
).	O
	O
We	O
next	O
used	O
smFRET	O
to	O
probe	O
the	O
conformational	O
selection	O
of	O
distinct	O
inter	O
-	O
RRM	O
arrangements	O
following	O
association	O
of	O
U2AF65	O
with	O
the	O
AdML	O
Py	O
-	O
tract	O
prototype	O
.	O
	O
To	O
assess	O
the	O
possible	O
contributions	O
of	O
RNA	O
-	O
free	O
conformations	O
of	O
U2AF65	O
and	O
/	O
or	O
structural	O
heterogeneity	O
introduced	O
by	O
tethering	O
of	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	O
/	O
Cy5	O
)	O
to	O
the	O
slide	O
to	O
the	O
observed	O
distribution	O
of	O
FRET	O
values	O
,	O
we	O
reversed	O
the	O
immobilization	O
scheme	O
.	O
	O
Therefore	O
,	O
RRM1	O
-	O
to	O
-	O
RRM2	O
distance	O
remains	O
similar	O
regardless	O
of	O
whether	O
U2AF65	O
is	O
bound	O
to	O
interrupted	O
or	O
continuous	O
Py	O
tract	O
.	O
	O
The	O
inter	O
-	O
fluorophore	O
distances	O
derived	O
from	O
the	O
observed	O
0	O
.	O
45	O
FRET	O
state	O
agree	O
with	O
the	O
distances	O
between	O
the	O
α	O
-	O
carbon	O
atoms	O
of	O
the	O
respective	O
residues	O
in	O
the	O
crystal	O
structures	O
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
bound	O
to	O
Py	O
-	O
tract	O
oligonucleotides	O
.	O
	O
Hidden	O
Markov	O
modelling	O
analysis	O
of	O
smFRET	O
traces	O
suggests	O
that	O
RNA	O
-	O
bound	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
can	O
sample	O
at	O
least	O
two	O
other	O
conformations	O
corresponding	O
to	O
∼	O
0	O
.	O
7	O
–	O
0	O
.	O
8	O
and	O
∼	O
0	O
.	O
3	O
FRET	O
values	O
in	O
addition	O
to	O
the	O
predominant	O
conformation	O
corresponding	O
to	O
the	O
0	O
.	O
45	O
FRET	O
state	O
.	O
	O
Truncation	O
of	O
U2AF65	O
to	O
the	O
core	O
RRM1	O
–	O
RRM2	O
region	O
reduces	O
its	O
RNA	O
affinity	O
by	O
100	O
-	O
fold	O
.	O
	O
As	O
such	O
,	O
we	O
suggest	O
that	O
the	O
MDS	O
-	O
relevant	O
U2AF65	O
mutations	O
contribute	O
to	O
MDS	O
progression	O
indirectly	O
,	O
by	O
destabilizing	O
a	O
relevant	O
conformation	O
of	O
the	O
conjoined	O
U2AF35	O
subunit	O
rather	O
than	O
affecting	O
U2AF65	O
functions	O
in	O
RNA	O
binding	O
or	O
spliceosome	O
recruitment	O
per	O
se	O
.	O
	O
An	O
increased	O
prevalence	O
of	O
the	O
∼	O
0	O
.	O
45	O
FRET	O
value	O
following	O
U2AF65	O
–	O
RNA	O
binding	O
,	O
coupled	O
with	O
the	O
apparent	O
absence	O
of	O
transitions	O
in	O
many	O
∼	O
0	O
.	O
45	O
-	O
value	O
single	O
molecule	O
traces	O
(	O
for	O
example	O
,	O
Fig	O
.	O
6e	O
),	O
suggests	O
a	O
population	O
shift	O
in	O
which	O
RNA	O
binds	O
to	O
(	O
and	O
draws	O
the	O
equilibrium	O
towards	O
)	O
a	O
pre	O
-	O
configured	O
inter	O
-	O
RRM	O
proximity	O
that	O
most	O
often	O
corresponds	O
to	O
the	O
∼	O
0	O
.	O
45	O
FRET	O
value	O
.	O
	O
Examples	O
of	O
‘	O
extended	O
conformational	O
selection	O
'	O
during	O
ligand	O
binding	O
have	O
been	O
characterized	O
for	O
a	O
growing	O
number	O
of	O
macromolecules	O
(	O
for	O
example	O
,	O
adenylate	O
kinase	O
,	O
LAO	O
-	O
binding	O
protein	O
,	O
poly	O
-	O
ubiquitin	O
,	O
maltose	O
-	O
binding	O
protein	O
and	O
the	O
preQ1	O
riboswitch	O
,	O
among	O
others	O
).	O
	O
These	O
transitions	O
could	O
correspond	O
to	O
rearrangement	O
from	O
the	O
‘	O
closed	O
'	O
NMR	O
/	O
PRE	O
-	O
based	O
U2AF65	O
conformation	O
in	O
which	O
the	O
RNA	O
-	O
binding	O
surface	O
of	O
only	O
a	O
single	O
RRM	O
is	O
exposed	O
and	O
available	O
for	O
RNA	O
binding	O
,	O
to	O
the	O
structural	O
state	O
seen	O
in	O
the	O
side	O
-	O
by	O
-	O
side	O
,	O
RNA	O
-	O
bound	O
crystal	O
structure	O
.	O
	O
The	O
finding	O
that	O
U2AF65	O
recognizes	O
a	O
nine	O
base	O
pair	O
Py	O
tract	O
contributes	O
to	O
an	O
elusive	O
‘	O
code	O
'	O
for	O
predicting	O
splicing	O
patterns	O
from	O
primary	O
sequences	O
in	O
the	O
post	O
-	O
genomic	O
era	O
(	O
reviewed	O
in	O
ref	O
.).	O
	O
(	O
b	O
)	O
Comparison	O
of	O
the	O
apparent	O
equilibrium	O
affinities	O
of	O
various	O
U2AF65	O
constructs	O
for	O
binding	O
the	O
prototypical	O
AdML	O
Py	O
tract	O
(	O
5	O
′-	O
CCCUUUUUUUUCC	O
-	O
3	O
′).	O
	O
The	O
apparent	O
equilibrium	O
dissociation	O
constants	O
(	O
KD	O
)	O
for	O
binding	O
the	O
AdML	O
13mer	O
are	O
as	O
follows	O
:	O
flU2AF65	O
,	O
30	O
±	O
3	O
nM	O
;	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
,	O
35	O
±	O
6	O
nM	O
;	O
U2AF651	B-mutant
,	I-mutant
2	I-mutant
,	O
3	O
,	O
600	O
±	O
300	O
nM	O
.	O
(	O
c	O
)	O
Comparison	O
of	O
the	O
RNA	O
sequence	O
specificities	O
of	O
flU2AF65	O
and	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
constructs	O
binding	O
C	O
-	O
rich	O
Py	O
tracts	O
with	O
4U	O
'	O
s	O
embedded	O
in	O
either	O
the	O
5	O
′-	O
(	O
light	O
grey	O
fill	O
)	O
or	O
3	O
′-	O
(	O
dark	O
grey	O
fill	O
)	O
regions	O
.	O
	O
The	O
purified	O
protein	O
and	O
average	O
fitted	O
fluorescence	O
anisotropy	O
RNA	O
-	O
binding	O
curves	O
are	O
shown	O
in	O
Supplementary	O
Fig	O
.	O
1	O
.	O
	O
(	O
b	O
)	O
Stereo	O
views	O
of	O
a	O
‘	O
kicked	O
'	O
2	O
|	O
Fo	O
|−|	O
Fc	O
|	O
electron	O
density	O
map	O
contoured	O
at	O
1σ	O
for	O
the	O
inter	O
-	O
RRM	O
linker	O
,	O
N	O
-	O
and	O
C	O
-	O
terminal	O
residues	O
(	O
blue	O
)	O
or	O
bound	O
oligonucleotide	O
of	O
a	O
representative	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
structure	O
(	O
structure	O
iv	O
,	O
bound	O
to	O
5	O
′-(	O
P	O
)	O
rUrUrUdUrUrU	O
(	O
BrdU	O
)	O
dUrC	O
)	O
(	O
magenta	O
).	O
	O
BrdU	O
,	O
5	O
-	O
bromo	O
-	O
deoxy	O
-	O
uridine	O
;	O
d	O
,	O
deoxy	O
-	O
ribose	O
;	O
P	O
-,	O
5	O
′-	O
phosphorylation	O
;	O
r	O
,	O
ribose	O
.	O
	O
The	O
U2AF65	O
linker	O
/	O
RRM	O
and	O
inter	O
-	O
RRM	O
interactions	O
.	O
	O
RNA	O
binding	O
stabilizes	O
the	O
side	O
-	O
by	O
-	O
side	O
conformation	O
of	O
U2AF65	O
RRMs	O
.	O
	O
Additional	O
traces	O
for	O
untethered	O
,	O
RNA	O
-	O
bound	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	O
/	O
Cy5	O
)	O
are	O
shown	O
in	O
Supplementary	O
Fig	O
.	O
7c	O
,	O
d	O
.	O
Histograms	O
(	O
d	O
,	O
f	O
,	O
h	O
,	O
j	O
)	O
show	O
the	O
distribution	O
of	O
FRET	O
values	O
in	O
RNA	O
-	O
free	O
,	O
slide	O
-	O
tethered	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	O
/	O
Cy5	O
)	O
(	O
d	O
);	O
AdML	O
RNA	O
-	O
bound	O
,	O
slide	O
-	O
tethered	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	O
/	O
Cy5	O
)	O
(	O
f	O
);	O
AdML	O
RNA	O
-	O
bound	O
,	O
untethered	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	O
/	O
Cy5	O
)	O
(	O
h	O
)	O
and	O
adenosine	O
-	O
interrupted	O
RNA	O
-	O
bound	O
,	O
slide	O
-	O
tethered	O
U2AF651	B-mutant
,	I-mutant
2LFRET	I-mutant
(	O
Cy3	O
/	O
Cy5	O
)	O
(	O
j	O
).	O
	O
A	O
surface	O
representation	O
of	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
is	O
shown	O
bound	O
to	O
nine	O
nucleotides	O
(	O
nt	O
);	O
the	O
relative	O
distances	O
and	O
juxtaposition	O
of	O
the	O
branch	O
point	O
sequence	O
(	O
BPS	O
)	O
and	O
consensus	O
AG	O
dinucleotide	O
at	O
the	O
3	O
′	O
splice	O
site	O
are	O
unknown	O
.	O
	O
(	O
b	O
)	O
Following	O
binding	O
to	O
the	O
Py	O
-	O
tract	O
RNA	O
,	O
a	O
conformation	O
corresponding	O
to	O
high	O
FRET	O
and	O
consistent	O
with	O
the	O
‘	O
closed	O
',	O
back	O
-	O
to	O
-	O
back	O
apo	O
-	O
U2AF65	O
model	O
resulting	O
from	O
PRE	O
/	O
NMR	O
characterization	O
(	O
PDB	O
ID	O
2YH0	O
)	O
often	O
transitions	O
to	O
a	O
conformation	O
corresponding	O
to	O
∼	O
0	O
.	O
45	O
FRET	O
value	O
,	O
which	O
is	O
consistent	O
with	O
‘	O
open	O
',	O
side	O
-	O
by	O
-	O
side	O
RRMs	O
such	O
as	O
the	O
U2AF651	B-mutant
,	I-mutant
2L	I-mutant
crystal	O
structures	O
.	O
	O
Over	O
a	O
large	O
dose	O
range	O
,	O
the	O
RNA	O
was	O
found	O
to	O
be	O
far	O
less	O
susceptible	O
to	O
radiation	O
-	O
induced	O
chemical	O
changes	O
than	O
the	O
protein	O
.	O
	O
Dose	O
is	O
defined	O
as	O
the	O
absorbed	O
energy	O
per	O
unit	O
mass	O
of	O
crystal	O
in	O
grays	O
(	O
Gy	O
;	O
1	O
Gy	O
=	O
1	O
J	O
kg	O
−	O
1	O
),	O
and	O
is	O
the	O
metric	O
against	O
which	O
damage	O
progression	O
should	O
be	O
monitored	O
during	O
MX	O
data	O
collection	O
,	O
as	O
opposed	O
to	O
time	O
.	O
	O
There	O
are	O
a	O
number	O
of	O
cases	O
where	O
SRD	O
manifestations	O
have	O
compromised	O
the	O
biological	O
information	O
extracted	O
from	O
MX	O
-	O
determined	O
structures	O
at	O
much	O
lower	O
doses	O
than	O
the	O
recommended	O
30	O
MGy	O
limit	O
,	O
leading	O
to	O
false	O
structural	O
interpretations	O
of	O
protein	O
mechanisms	O
.	O
	O
The	O
investigation	O
of	O
naturally	O
forming	O
nucleoprotein	O
complexes	O
circumvents	O
the	O
inherent	O
challenges	O
in	O
making	O
controlled	O
comparisons	O
of	O
damage	O
mechanisms	O
between	O
protein	O
and	O
nucleic	O
acids	O
crystallized	O
separately	O
.	O
Recently	O
,	O
for	O
a	O
well	O
characterized	O
bacterial	O
protein	O
–	O
DNA	O
complex	O
(	O
C	O
.	O
Esp1396I	O
;	O
PDB	O
entry	O
3clc	O
;	O
resolution	O
2	O
.	O
8	O
Å	O
;	O
McGeehan	O
et	O
al	O
.,	O
2008	O
)	O
it	O
was	O
concluded	O
that	O
over	O
a	O
wide	O
dose	O
range	O
(	O
2	O
.	O
1	O
–	O
44	O
.	O
6	O
MGy	O
)	O
the	O
protein	O
was	O
far	O
more	O
susceptible	O
to	O
SRD	O
than	O
the	O
DNA	O
within	O
the	O
crystal	O
(	O
Bury	O
et	O
al	O
.,	O
2015	O
).	O
	O
Three	O
acidic	O
residues	O
(	O
Glu36	O
,	O
Asp39	O
and	O
Glu42	O
)	O
are	O
involved	O
in	O
RNA	O
interactions	O
within	O
each	O
of	O
the	O
11	O
TRAP	O
ring	O
subunits	O
,	O
and	O
Fig	O
.	O
5	O
▸	O
shows	O
their	O
density	O
changes	O
with	O
increasing	O
dose	O
.	O
	O
Salt	O
-	O
bridge	O
interactions	O
have	O
previously	O
been	O
suggested	O
to	O
reduce	O
the	O
glutamate	O
decarboxylation	O
rate	O
within	O
the	O
large	O
(∼	O
62	O
.	O
4	O
kDa	O
)	O
myrosinase	O
protein	O
structure	O
(	O
Burmeister	O
,	O
2000	O
).	O
	O
The	O
extended	O
aliphatic	O
Lys37	O
side	O
chain	O
stacks	O
against	O
the	O
nearby	O
G1	O
base	O
,	O
making	O
a	O
series	O
of	O
nonpolar	O
contacts	O
within	O
each	O
RNA	O
-	O
binding	O
interface	O
.	O
	O
Representative	O
Phe32	O
and	O
Lys37	O
atoms	O
were	O
selected	O
to	O
illustrate	O
these	O
trends	O
.	O
	O
Our	O
method	O
­	O
ology	O
,	O
which	O
eliminated	O
tedious	O
and	O
error	O
-	O
prone	O
visual	O
inspection	O
,	O
permitted	O
the	O
determination	O
on	O
a	O
per	O
-	O
atom	O
basis	O
of	O
the	O
most	O
damaged	O
sites	O
,	O
as	O
characterized	O
by	O
F	O
obs	O
(	O
d	O
n	O
)	O
−	O
F	O
obs	O
(	O
d	O
1	O
)	O
Fourier	O
difference	O
map	O
peaks	O
between	O
successive	O
data	O
sets	O
collected	O
from	O
the	O
same	O
crystal	O
.	O
	O
Both	O
Glu36	O
and	O
Asp39	O
bind	O
directly	O
to	O
RNA	O
,	O
each	O
through	O
two	O
hydrogen	O
bonds	O
to	O
guanine	O
bases	O
(	O
G3	O
and	O
G1	O
,	O
respectively	O
).	O
	O
Observations	O
of	O
lower	O
protein	O
radiation	O
-	O
sensitivity	O
in	O
DNA	O
-	O
bound	O
forms	O
have	O
been	O
recorded	O
in	O
solution	O
at	O
RT	O
at	O
much	O
lower	O
doses	O
(∼	O
1	O
kGy	O
)	O
than	O
those	O
used	O
for	O
typical	O
MX	O
experiments	O
[	O
e	O
.	O
g	O
.	O
an	O
oestrogen	O
response	O
element	O
–	O
receptor	O
complex	O
(	O
Stísová	O
et	O
al	O
.,	O
2006	O
)	O
and	O
a	O
DNA	O
glycosylase	O
and	O
its	O
abasic	O
DNA	O
target	O
site	O
(	O
Gillard	O
et	O
al	O
.,	O
2004	O
)].	O
	O
However	O
,	O
in	O
the	O
current	O
MX	O
study	O
at	O
100	O
K	O
,	O
the	O
main	O
damaging	O
species	O
are	O
believed	O
to	O
be	O
migrating	O
LEEs	O
and	O
holes	O
produced	O
directly	O
within	O
the	O
protein	O
–	O
RNA	O
components	O
or	O
in	O
closely	O
associated	O
solvent	O
.	O
	O
RNA	O
is	O
shown	O
is	O
yellow	O
.	O
	O
(	O
b	O
)	O
Average	O
D	O
loss	O
for	O
each	O
residue	O
/	O
nucleotide	O
type	O
with	O
respect	O
to	O
the	O
DWD	O
(	O
diffraction	O
-	O
weighted	O
dose	O
;	O
Zeldin	O
,	O
Brock	O
­	O
hauser	O
et	O
al	O
.,	O
2013	O
).	O
	O
Residues	O
have	O
been	O
grouped	O
by	O
amino	O
-	O
acid	O
number	O
,	O
and	O
split	O
into	O
bound	O
and	O
nonbound	O
groupings	O
,	O
with	O
each	O
bar	O
representing	O
the	O
mean	O
calculated	O
over	O
11	O
equivalent	O
atoms	O
around	O
a	O
TRAP	O
ring	O
.	O
	O
The	O
three	O
best	O
-	O
characterized	O
MAPK	O
signalling	O
pathways	O
are	O
mediated	O
by	O
the	O
kinases	O
extracellular	O
signal	O
-	O
regulated	O
kinase	O
(	O
ERK	O
),	O
c	O
-	O
Jun	O
N	O
-	O
terminal	O
kinase	O
(	O
JNK	O
)	O
and	O
p38	O
.	O
	O
The	O
ERK	O
pathway	O
is	O
activated	O
by	O
various	O
mitogens	O
and	O
phorbol	O
esters	O
,	O
whereas	O
the	O
JNK	O
and	O
p38	O
pathways	O
are	O
stimulated	O
mainly	O
by	O
environmental	O
stress	O
and	O
inflammatory	O
cytokines	O
.	O
	O
MKPs	O
constitute	O
a	O
group	O
of	O
DUSPs	O
that	O
are	O
characterized	O
by	O
their	O
ability	O
to	O
dephosphorylate	O
both	O
phosphotyrosine	O
and	O
phosphoserine	O
/	O
phospho	O
-	O
threonine	O
residues	O
within	O
a	O
substrate	O
.	O
	O
Biochemical	O
and	O
modelling	O
studies	O
further	O
demonstrate	O
that	O
the	O
molecular	O
interactions	O
mediate	O
this	O
key	O
element	O
for	O
substrate	O
recognition	O
are	O
highly	O
conserved	O
among	O
all	O
MKP	O
-	O
family	O
members	O
.	O
	O
In	O
mammalian	O
cells	O
,	O
the	O
MKP	O
subfamily	O
includes	O
10	O
distinct	O
catalytically	O
active	O
MKPs	O
.	O
	O
Figure	O
2b	O
shows	O
the	O
variation	O
of	O
initial	O
rates	O
of	O
the	O
MKP7ΔC304	B-mutant
and	O
MKP7	O
-	O
CD	O
-	O
catalysed	O
reaction	O
with	O
the	O
concentration	O
of	O
phospho	O
-	O
JNK1	O
.	O
Because	O
the	O
concentrations	O
of	O
MKP7	O
and	O
pJNK1	O
were	O
comparable	O
in	O
the	O
reaction	O
,	O
the	O
assumption	O
that	O
the	O
free	O
-	O
substrate	O
concentration	O
is	O
equal	O
to	O
the	O
total	O
substrate	O
concentration	O
is	O
not	O
valid	O
.	O
	O
To	O
further	O
confirm	O
the	O
JNK1	O
–	O
MKP7	O
-	O
CD	O
interaction	O
,	O
we	O
performed	O
a	O
pull	O
-	O
down	O
assay	O
using	O
the	O
purified	O
proteins	O
.	O
	O
The	O
catalytic	O
domain	O
of	O
MKP7	O
interacts	O
with	O
JNK1	O
through	O
a	O
contiguous	O
surface	O
area	O
that	O
is	O
remote	O
from	O
the	O
active	O
site	O
.	O
	O
The	O
active	O
site	O
of	O
MKP7	O
consists	O
of	O
the	O
phosphate	O
-	O
binding	O
loop	O
(	O
P	O
-	O
loop	O
,	O
Cys244	O
-	O
Leu245	O
-	O
Ala246	O
-	O
Gly247	O
-	O
Ile248	O
-	O
Ser249	O
-	O
Arg250	O
),	O
and	O
Asp213	O
in	O
the	O
general	O
acid	O
loop	O
(	O
Fig	O
.	O
3b	O
and	O
Supplementary	O
Fig	O
.	O
1b	O
).	O
	O
The	O
side	O
chain	O
of	O
strictly	O
conserved	O
Arg250	O
is	O
oriented	O
towards	O
the	O
negatively	O
charged	O
chloride	O
,	O
similar	O
to	O
the	O
canonical	O
phosphate	O
-	O
coordinating	O
conformation	O
.	O
	O
In	O
the	O
complex	O
,	O
MKP7	O
-	O
CD	O
and	O
JNK1	O
form	O
extensive	O
protein	O
–	O
protein	O
interactions	O
involving	O
the	O
C	O
-	O
terminal	O
helices	O
of	O
MKP7	O
-	O
CD	O
and	O
C	O
-	O
lobe	O
of	O
JNK1	O
(	O
Fig	O
.	O
3d	O
,	O
e	O
).	O
	O
Mutation	O
of	O
Leu288	O
markedly	O
reduced	O
its	O
solubility	O
when	O
expressed	O
in	O
Escherichia	O
coli	O
,	O
resulting	O
in	O
the	O
insoluble	O
aggregation	O
of	O
the	O
mutant	O
protein	O
.	O
	O
The	O
small	O
pNPP	O
molecule	O
binds	O
directly	O
at	O
the	O
enzyme	O
active	O
site	O
and	O
can	O
be	O
used	O
to	O
probe	O
the	O
reaction	O
mechanism	O
of	O
protein	O
phosphatases	O
.	O
	O
Biochemical	O
results	O
suggested	O
that	O
the	O
affinity	O
and	O
specificity	O
between	O
KAP	O
and	O
CDK2	O
results	O
from	O
the	O
recognition	O
site	O
comprising	O
CDK2	O
residues	O
from	O
the	O
αG	O
helix	O
and	O
L14	O
loop	O
and	O
the	O
N	O
-	O
terminal	O
helical	O
region	O
of	O
KAP	O
(	O
Fig	O
.	O
5b	O
).	O
	O
Structural	O
analysis	O
and	O
sequence	O
alignment	O
reveal	O
that	O
one	O
of	O
the	O
few	O
differences	O
between	O
MKP7	O
-	O
CD	O
and	O
KAP	O
in	O
the	O
substrate	O
-	O
binding	O
region	O
is	O
the	O
presence	O
of	O
the	O
motif	O
FNFL	O
in	O
MKP7	O
-	O
CD	O
,	O
which	O
corresponds	O
to	O
IKQY	O
in	O
KAP	O
(	O
Fig	O
.	O
5c	O
).	O
	O
Parallel	O
experiments	O
showed	O
clearly	O
that	O
the	O
D	O
-	O
motif	O
mutants	O
(	O
R56A	B-mutant
/	O
R57A	B-mutant
and	O
V63A	B-mutant
/	O
I65A	B-mutant
)	O
dephosphorylated	O
JNK	O
as	O
did	O
the	O
wild	O
type	O
under	O
the	O
same	O
conditions	O
,	O
further	O
confirming	O
that	O
the	O
MKP7	O
-	O
KBD	O
is	O
not	O
required	O
for	O
the	O
JNK	O
inactivation	O
in	O
vivo	O
.	O
	O
Consistent	O
with	O
the	O
in	O
vitro	O
data	O
,	O
the	O
level	O
of	O
phosphorylated	O
JNK	O
was	O
not	O
or	O
little	O
altered	O
in	O
MKP7	O
FXF	O
-	O
motif	O
mutants	O
(	O
F285D	B-mutant
,	O
F287D	B-mutant
and	O
L288D	B-mutant
)-	O
transfected	O
cells	O
,	O
and	O
the	O
MKP7	O
D268A	B-mutant
and	O
N286A	B-mutant
mutants	O
retained	O
the	O
ability	O
to	O
reduce	O
the	O
phosphorylation	O
levels	O
of	O
JNK	O
.	O
	O
In	O
agreement	O
with	O
the	O
in	O
vitro	O
pull	O
-	O
down	O
results	O
,	O
the	O
mutants	O
D229A	B-mutant
,	O
W234D	B-mutant
and	O
Y259D	B-mutant
were	O
not	O
co	O
-	O
precipitated	O
with	O
MKP7	O
,	O
and	O
the	O
I231D	B-mutant
mutant	O
had	O
only	O
little	O
effect	O
on	O
the	O
JNK1	O
–	O
MKP7	O
interaction	O
(	O
Fig	O
.	O
6d	O
and	O
Supplementary	O
Fig	O
.	O
3a	O
).	O
	O
Moreover	O
,	O
treatment	O
of	O
cells	O
expressing	O
MKP7	O
-	O
KBD	O
mutants	O
(	O
R56A	B-mutant
/	O
R57A	B-mutant
and	O
V63A	B-mutant
/	O
I65A	B-mutant
)	O
decreased	O
the	O
apoptosis	O
rates	O
to	O
a	O
similar	O
extent	O
as	O
MKP7	O
wild	O
type	O
did	O
.	O
	O
MKP5	O
belongs	O
to	O
the	O
same	O
subfamily	O
as	O
MKP7	O
.	O
	O
In	O
contrast	O
to	O
p38α	O
substrate	O
,	O
deletion	O
of	O
the	O
MKP5	O
-	O
KBD	O
had	O
little	O
effects	O
on	O
the	O
kinetic	O
parameters	O
for	O
the	O
JNK1	O
dephosphorylation	O
,	O
indicating	O
that	O
the	O
KBD	O
of	O
MKP5	O
is	O
not	O
required	O
for	O
the	O
JNK1	O
dephosphorylation	O
(	O
Fig	O
.	O
7b	O
).	O
	O
As	O
shown	O
in	O
Fig	O
.	O
7f	O
,	O
the	O
T432A	B-mutant
and	O
L449F	B-mutant
MKP5	O
mutant	O
showed	O
little	O
or	O
no	O
difference	O
in	O
phosphatase	O
activity	O
,	O
whereas	O
the	O
other	O
mutants	O
showed	O
reduced	O
specific	O
activities	O
of	O
MKP5	O
.	O
	O
As	O
in	O
the	O
case	O
of	O
MKP7	O
,	O
all	O
the	O
mutants	O
,	O
except	O
F451D	B-mutant
/	I-mutant
A	I-mutant
,	O
showed	O
no	O
pNPPase	O
activity	O
changes	O
compared	O
with	O
the	O
wild	O
-	O
type	O
MKP5	O
-	O
CD	O
(	O
Fig	O
.	O
7g	O
),	O
and	O
the	O
point	O
mutations	O
in	O
JNK1	O
also	O
reduced	O
the	O
binding	O
affinity	O
of	O
MKP5	O
-	O
CD	O
for	O
JNK1	O
(	O
Fig	O
.	O
7h	O
).	O
	O
This	O
is	O
consistent	O
with	O
the	O
experimental	O
observation	O
showing	O
that	O
JNK1	O
binds	O
to	O
MKP7	O
-	O
CD	O
much	O
more	O
tightly	O
than	O
MKP5	O
-	O
CD	O
(	O
Km	O
value	O
of	O
MKP5	O
-	O
CD	O
for	O
pJNK1	O
substrate	O
is	O
∼	O
20	O
-	O
fold	O
higher	O
than	O
that	O
of	O
MKP7	O
-	O
CD	O
).	O
	O
The	O
MAPKs	O
p38	O
,	O
ERK	O
and	O
JNK	O
,	O
are	O
central	O
to	O
evolutionarily	O
conserved	O
signalling	O
pathways	O
that	O
are	O
present	O
in	O
all	O
eukaryotic	O
cells	O
.	O
	O
Each	O
MAPK	O
cascade	O
is	O
activated	O
in	O
response	O
to	O
a	O
diverse	O
array	O
of	O
extracellular	O
signals	O
and	O
culminates	O
in	O
the	O
dual	O
-	O
phosphorylation	O
of	O
a	O
threonine	O
and	O
a	O
tyrosine	O
residue	O
in	O
the	O
MAPK	O
-	O
activation	O
loop	O
.	O
	O
This	O
structure	O
reveals	O
an	O
FXF	O
-	O
docking	O
interaction	O
mode	O
between	O
MAPK	O
and	O
MKP	O
.	O
	O
When	O
MKP7	O
is	O
bound	O
to	O
JIP	O
-	O
1	O
,	O
it	O
reduces	O
JNK	O
activation	O
,	O
leading	O
to	O
reduced	O
phosphorylation	O
of	O
the	O
JNK	O
target	O
c	O
-	O
Jun	O
.	O
	O
The	O
colour	O
scheme	O
is	O
the	O
same	O
in	O
the	O
following	O
figures	O
unless	O
indicated	O
otherwise	O
.	O
(	O
b	O
)	O
Plots	O
of	O
initial	O
velocity	O
of	O
the	O
MKP7	O
-	O
catalysed	O
reaction	O
versus	O
phospho	O
-	O
JNK1	O
concentration	O
.	O
	O
The	O
top	O
panel	O
shows	O
the	O
relative	O
affinities	O
of	O
MKP7	O
-	O
CD	O
and	O
MKP7	O
-	O
KBD	O
to	O
JNK1	O
,	O
with	O
the	O
affinity	O
of	O
MKP7	O
-	O
CD	O
defined	O
as	O
100	O
%;	O
the	O
middle	O
panel	O
is	O
the	O
electrophoretic	O
pattern	O
of	O
MKP7	O
and	O
JNK1	O
after	O
GST	O
pull	O
-	O
down	O
assays	O
.	O
	O
Blue	O
dashed	O
lines	O
represent	O
polar	O
interactions	O
.	O
	O
The	O
CDK2	O
is	O
shown	O
in	O
surface	O
representation	O
coloured	O
according	O
to	O
the	O
electrostatic	O
potential	O
(	O
positive	O
,	O
blue	O
;	O
negative	O
,	O
red	O
).	O
	O
Residues	O
of	O
MKP7	O
-	O
CD	O
involved	O
in	O
JNK1	O
recognition	O
are	O
indicated	O
by	O
cyan	O
asterisks	O
,	O
and	O
the	O
conserved	O
FXF	O
-	O
motif	O
is	O
highlighted	O
in	O
cyan	O
.	O
	O
The	O
secondary	O
structure	O
assignments	O
of	O
MKP7	O
-	O
CD	O
and	O
KAP	O
are	O
shown	O
above	O
and	O
below	O
each	O
sequence	O
.	O
	O
Shown	O
is	O
a	O
typical	O
immunoblot	O
for	O
phosphorylated	O
JNK	O
from	O
three	O
independent	O
experiments	O
.	O
	O
The	O
results	O
using	O
Annexin	O
-	O
V	O
stain	O
for	O
membrane	O
phosphatidylserine	O
eversion	O
,	O
combined	O
with	O
propidium	O
iodide	O
(	O
PI	O
)	O
uptake	O
to	O
evaluate	O
cells	O
whose	O
membranes	O
had	O
been	O
compromised	O
.	O
	O
The	O
solid	O
lines	O
are	O
best	O
-	O
fitting	O
results	O
according	O
to	O
the	O
Michaelis	O
–	O
Menten	O
equation	O
with	O
Km	O
and	O
kcat	O
values	O
indicated	O
.	O
	O
(	O
d	O
)	O
Gel	O
filtration	O
analysis	O
for	O
interaction	O
of	O
JNK1	O
with	O
MKP5	O
-	O
CD	O
and	O
MKP5	O
-	O
KBD	O
.	O
(	O
e	O
)	O
GST	O
-	O
mediated	O
pull	O
-	O
down	O
assays	O
for	O
interaction	O
of	O
JNK1	O
with	O
MKP5	O
-	O
CD	O
and	O
MKP5	O
-	O
KBD	O
.	O
	O
The	O
panels	O
are	O
arranged	O
the	O
same	O
as	O
in	O
Fig	O
.	O
2d	O
.	O
(	O
f	O
)	O
Effects	O
of	O
mutations	O
in	O
MKP5	O
-	O
CD	O
on	O
the	O
JNK1	O
dephosphorylation	O
(	O
mean	O
±	O
s	O
.	O
e	O
.	O
m	O
.,	O
n	O
=	O
3	O
).	O
	O
(	O
g	O
)	O
Effects	O
of	O
mutations	O
in	O
MKP5	O
-	O
CD	O
on	O
the	O
pNPP	O
hydrolysis	O
reaction	O
(	O
mean	O
±	O
s	O
.	O
e	O
.	O
m	O
.,	O
n	O
=	O
3	O
).	O
	O
The	O
HAESA	O
ectodomain	O
folds	O
into	O
a	O
superhelical	O
assembly	O
of	O
21	O
leucine	O
-	O
rich	O
repeats	O
.	O
	O
The	O
HAESA	O
ectodomain	O
is	O
shown	O
in	O
blue	O
(	O
in	O
surface	O
representation	O
),	O
the	O
glycan	O
structures	O
are	O
shown	O
in	O
yellow	O
.	O
	O
Residues	O
mediating	O
hydrophobic	O
interactions	O
with	O
the	O
IDA	O
peptide	O
are	O
highlighted	O
in	O
blue	O
,	O
residues	O
contributing	O
to	O
hydrogen	O
bond	O
interactions	O
and	O
/	O
or	O
salt	O
bridges	O
are	O
shown	O
in	O
red	O
.	O
	O
The	O
alignment	O
includes	O
a	O
secondary	O
structure	O
assignment	O
calculated	O
with	O
the	O
program	O
DSSP	O
and	O
colored	O
according	O
to	O
Figure	O
1	O
,	O
with	O
the	O
N	O
-	O
and	O
C	O
-	O
terminal	O
caps	O
and	O
the	O
21	O
LRR	O
motifs	O
indicated	O
in	O
orange	O
and	O
blue	O
,	O
respectively	O
.	O
	O
Void	O
(	O
V0	O
)	O
volume	O
and	O
total	O
volume	O
(	O
Vt	O
)	O
are	O
shown	O
,	O
together	O
with	O
elution	O
volumes	O
for	O
molecular	O
mass	O
standards	O
(	O
A	O
,	O
Thyroglobulin	O
,	O
669	O
,	O
000	O
Da	O
;	O
B	O
,	O
Ferritin	O
,	O
440	O
,	O
00	O
Da	O
,	O
C	O
,	O
Aldolase	O
,	O
158	O
,	O
000	O
Da	O
;	O
D	O
,	O
Conalbumin	O
,	O
75	O
,	O
000	O
Da	O
;	O
E	O
,	O
Ovalbumin	O
,	O
44	O
,	O
000	O
Da	O
;	O
F	O
,	O
Carbonic	O
anhydrase	O
,	O
29	O
,	O
000	O
Da	O
).	O
	O
Mutant	O
(	O
m	O
)	O
versions	O
,	O
which	O
carry	O
point	O
mutations	O
in	O
their	O
active	O
sites	O
(	O
Asp837HAESA	B-mutant
→	I-mutant
Asn	I-mutant
,	O
Asp447SERK1	B-mutant
→	I-mutant
Asn	I-mutant
)	O
possess	O
no	O
autophosphorylation	O
activity	O
(	O
lanes	O
2	O
+	O
4	O
).	O
	O
We	O
next	O
determined	O
crystal	O
structures	O
of	O
the	O
apo	O
HAESA	O
ectodomain	O
and	O
of	O
a	O
HAESA	O
-	O
IDA	O
complex	O
,	O
at	O
1	O
.	O
74	O
and	O
1	O
.	O
86	O
Å	O
resolution	O
,	O
respectively	O
(	O
Figure	O
1C	O
;	O
Figure	O
1	O
—	O
figure	O
supplement	O
1B	O
–	O
D	O
;	O
Tables	O
1	O
,	O
2	O
).	O
	O
We	O
next	O
tested	O
if	O
HAESA	O
binds	O
other	O
IDA	O
peptide	O
family	O
members	O
.	O
	O
Notably	O
,	O
HAESA	O
can	O
discriminate	O
between	O
IDLs	O
and	O
functionally	O
unrelated	O
dodecamer	O
peptides	O
with	O
Hyp	O
modifications	O
,	O
such	O
as	O
CLV3	O
(	O
Figures	O
2D	O
,	O
7	O
).	O
	O
Our	O
experiments	O
suggest	O
that	O
among	O
the	O
SERK	O
family	O
members	O
,	O
SERK1	O
is	O
a	O
positive	O
regulator	O
of	O
floral	O
abscission	O
.	O
	O
We	O
thus	O
focused	O
on	O
analyzing	O
the	O
contribution	O
of	O
SERK1	O
to	O
HAESA	O
ligand	O
sensing	O
and	O
receptor	O
activation	O
.	O
	O
Our	O
calorimetry	O
experiments	O
now	O
reveal	O
that	O
SERKs	O
may	O
render	O
HAESA	O
,	O
and	O
potentially	O
other	O
receptor	O
kinases	O
,	O
competent	O
for	O
high	O
-	O
affinity	O
sensing	O
of	O
their	O
cognate	O
ligands	O
.	O
	O
Together	O
,	O
our	O
genetic	O
and	O
biochemical	O
experiments	O
implicate	O
SERK1	O
as	O
a	O
HAESA	O
co	O
-	O
receptor	O
in	O
the	O
Arabidopsis	O
abscission	O
zone	O
.	O
	O
The	O
conformational	O
change	O
in	O
the	O
C	O
-	O
terminal	O
LRRs	O
and	O
capping	O
domain	O
is	O
indicated	O
by	O
an	O
arrow	O
.	O
(	O
C	O
)	O
SERK1	O
forms	O
an	O
integral	O
part	O
of	O
the	O
receptor	O
'	O
s	O
peptide	O
binding	O
pocket	O
.	O
	O
The	O
SERK1	O
ectodomain	O
interacts	O
with	O
the	O
IDA	O
peptide	O
binding	O
site	O
using	O
a	O
loop	O
region	O
(	O
residues	O
51	O
-	O
59SERK1	O
)	O
from	O
its	O
N	O
-	O
terminal	O
cap	O
(	O
Figure	O
4A	O
,	O
C	O
).	O
	O
Deletion	O
of	O
the	O
C	O
-	O
terminal	O
Asn69IDA	O
completely	O
inhibits	O
complex	O
formation	O
.	O
	O
15	O
out	O
of	O
15	O
35S	O
::	O
IDA	O
plants	O
,	O
0	O
out	O
of	O
15	O
Col	O
-	O
0	O
plants	O
and	O
0	O
out	O
of	O
15	O
35S	O
::	O
IDA	B-mutant
K66A	I-mutant
/	I-mutant
R67A	I-mutant
double	O
-	O
mutant	O
plants	O
,	O
showed	O
an	O
enlarged	O
abscission	O
zone	O
,	O
respectively	O
(	O
3	O
independent	O
lines	O
were	O
analyzed	O
).	O
	O
In	O
contrast	O
,	O
over	O
-	O
expression	O
of	O
the	O
IDA	B-mutant
Lys66IDA	I-mutant
/	I-mutant
Arg67IDA	I-mutant
→	I-mutant
Ala	I-mutant
double	O
mutant	O
significantly	O
delays	O
floral	O
abscission	O
when	O
compared	O
to	O
wild	O
-	O
type	O
control	O
plants	O
,	O
suggesting	O
that	O
the	O
mutant	O
IDA	O
peptide	O
has	O
reduced	O
activity	O
in	O
planta	O
(	O
Figure	O
5C	O
–	O
E	O
).	O
	O
For	O
a	O
rapidly	O
growing	O
number	O
of	O
plant	O
signaling	O
pathways	O
,	O
SERK	O
proteins	O
act	O
as	O
these	O
essential	O
co	O
-	O
receptors	O
(;	O
).	O
	O
The	O
central	O
Hyp	O
residue	O
in	O
IDA	O
is	O
found	O
buried	O
in	O
the	O
HAESA	O
peptide	O
binding	O
surface	O
and	O
thus	O
this	O
post	O
-	O
translational	O
modification	O
may	O
regulate	O
IDA	O
bioactivity	O
.	O
	O
In	O
our	O
quantitative	O
biochemical	O
assays	O
,	O
the	O
presence	O
of	O
SERK1	O
dramatically	O
increases	O
the	O
HAESA	O
binding	O
specificity	O
and	O
affinity	O
for	O
IDA	O
.	O
	O
It	O
is	O
of	O
note	O
that	O
our	O
reported	O
binding	O
affinities	O
for	O
IDA	O
and	O
SERK1	O
have	O
been	O
measured	O
using	O
synthetic	O
peptides	O
and	O
the	O
isolated	O
HAESA	O
and	O
SERK1	O
ectodomains	O
,	O
and	O
thus	O
might	O
differ	O
in	O
the	O
context	O
of	O
the	O
full	O
-	O
length	O
,	O
membrane	O
-	O
embedded	O
signaling	O
complex	O
.	O
	O
(	O
B	O
)	O
View	O
of	O
the	O
inner	O
surface	O
of	O
the	O
SERK1	O
LRR	O
domain	O
(	O
PDB	O
-	O
ID	O
4lsc	O
,	O
surface	O
representation	O
,	O
in	O
gray	O
).	O
	O
Structure	O
-	O
guided	O
multiple	O
sequence	O
alignment	O
of	O
IDA	O
and	O
IDA	O
-	O
like	O
peptides	O
with	O
other	O
plant	O
peptide	O
hormone	O
families	O
,	O
including	O
CLAVATA3	O
–	O
EMBRYO	O
SURROUNDING	O
REGION	O
-	O
RELATED	O
(	O
CLV3	O
/	O
CLE	O
),	O
ROOT	O
GROWTH	O
FACTOR	O
–	O
GOLVEN	O
(	O
RGF	O
/	O
GLV	O
),	O
PRECURSOR	O
GENE	O
PROPEP1	O
(	O
PEP1	O
)	O
from	O
Arabidopsis	O
thaliana	O
.	O
	O
It	O
is	O
interesting	O
to	O
note	O
,	O
that	O
CLEs	O
in	O
their	O
mature	O
form	O
are	O
also	O
hydroxyprolinated	O
dodecamers	O
,	O
which	O
bind	O
to	O
a	O
surface	O
area	O
in	O
the	O
BARELY	O
ANY	O
MERISTEM	O
1	O
receptor	O
that	O
would	O
correspond	O
to	O
part	O
of	O
the	O
IDA	O
binding	O
cleft	O
in	O
HAESA	O
.	O
	O
The	O
structures	O
suggest	O
a	O
trajectory	O
of	O
IRES	O
translocation	O
,	O
required	O
for	O
translation	O
initiation	O
,	O
and	O
provide	O
an	O
unprecedented	O
view	O
of	O
eEF2	O
dynamics	O
.	O
	O
To	O
initiate	O
translation	O
,	O
a	O
structured	O
IRES	O
RNA	O
interacts	O
with	O
the	O
40S	O
subunit	O
or	O
the	O
80S	O
ribosome	O
,	O
resulting	O
in	O
precise	O
positioning	O
of	O
the	O
downstream	O
start	O
codon	O
in	O
the	O
small	O
40S	O
subunit	O
.	O
	O
The	O
canonical	O
scenario	O
of	O
cap	O
-	O
dependent	O
and	O
IRES	O
-	O
dependent	O
initiation	O
involves	O
positioning	O
of	O
the	O
AUG	O
start	O
codon	O
and	O
the	O
initiator	O
tRNAMet	O
in	O
the	O
ribosomal	O
peptidyl	O
-	O
tRNA	O
(	O
P	O
)	O
site	O
,	O
facilitated	O
by	O
interaction	O
with	O
initiation	O
factors	O
.	O
	O
The	O
codon	O
-	O
anticodon	O
-	O
like	O
helix	O
of	O
PKI	O
is	O
stabilized	O
by	O
interactions	O
with	O
the	O
universally	O
conserved	O
decoding	O
-	O
center	O
nucleotides	O
G577	O
,	O
A1755	O
and	O
A1756	O
(	O
G530	O
,	O
A1492	O
and	O
A1493	O
in	O
E	O
.	O
coli	O
16S	O
ribosomal	O
RNA	O
,	O
or	O
rRNA	O
).	O
	O
How	O
this	O
non	O
-	O
canonical	O
initiation	O
complex	O
transitions	O
to	O
the	O
elongation	O
step	O
is	O
not	O
fully	O
understood	O
.	O
	O
Translocation	O
of	O
2tRNA	O
•	O
mRNA	O
involves	O
two	O
major	O
large	O
-	O
scale	O
ribosome	O
rearrangements	O
(	O
Figure	O
1	O
—	O
figure	O
supplement	O
1	O
)	O
(	O
reviewed	O
in	O
).	O
	O
Concurrently	O
,	O
the	O
deacyl	O
-	O
tRNA	O
interacts	O
with	O
the	O
P	O
site	O
of	O
the	O
small	O
subunit	O
and	O
the	O
E	O
site	O
of	O
the	O
large	O
subunit	O
(	O
P	O
/	O
E	O
hybrid	O
state	O
).	O
	O
Binding	O
of	O
EF	O
-	O
G	O
next	O
to	O
the	O
A	O
site	O
and	O
reverse	O
rotation	O
of	O
the	O
small	O
subunit	O
results	O
in	O
translocation	O
of	O
both	O
ASLs	O
on	O
the	O
small	O
subunit	O
.	O
	O
Structures	O
of	O
the	O
70S	O
•	O
EF	O
-	O
G	O
complex	O
bound	O
with	O
two	O
nearly	O
translocated	O
tRNAs	O
,	O
exhibit	O
a	O
large	O
18	O
°	O
to	O
21	O
°	O
head	O
swivel	O
in	O
a	O
mid	O
-	O
rotated	O
subunit	O
,	O
whereas	O
no	O
head	O
swivel	O
is	O
observed	O
in	O
the	O
fully	O
rotated	O
pre	O
-	O
translocation	O
or	O
in	O
the	O
non	O
-	O
rotated	O
post	O
-	O
translocation	O
70S	O
•	O
2tRNA	O
•	O
EF	O
-	O
G	O
structures	O
.	O
	O
The	O
head	O
swivel	O
was	O
proposed	O
to	O
facilitate	O
transition	O
of	O
the	O
tRNA	O
from	O
the	O
P	O
to	O
E	O
site	O
by	O
widening	O
a	O
constriction	O
between	O
these	O
sites	O
on	O
the	O
30S	O
subunit	O
.	O
	O
This	O
widening	O
allows	O
the	O
ASL	O
to	O
sample	O
positions	O
between	O
the	O
P	O
and	O
E	O
sites	O
.	O
	O
(	O
a	O
)	O
Structures	O
of	O
bacterial	O
70S	O
•	O
2tRNA	O
•	O
mRNA	O
translocation	O
complexes	O
,	O
ordered	O
according	O
to	O
the	O
position	O
of	O
the	O
translocating	O
A	O
->	O
P	O
tRNA	O
(	O
orange	O
).	O
	O
The	O
large	O
ribosomal	O
subunit	O
is	O
shown	O
in	O
cyan	O
;	O
the	O
small	O
subunit	O
in	O
light	O
yellow	O
(	O
head	O
)	O
and	O
wheat	O
-	O
yellow	O
(	O
body	O
),	O
elongation	O
factor	O
G	O
(	O
EF	O
-	O
G	O
)	O
is	O
shown	O
in	O
green	O
.	O
	O
Nucleotides	O
C1274	O
,	O
U1191	O
of	O
the	O
40S	O
head	O
and	O
G904	O
of	O
the	O
platform	O
(	O
corresponding	O
to	O
C1054	O
,	O
G966	O
and	O
G693	O
in	O
E	O
.	O
coli	O
16S	O
rRNA	O
)	O
are	O
shown	O
in	O
black	O
to	O
denote	O
the	O
A	O
,	O
P	O
and	O
E	O
sites	O
,	O
respectively	O
.	O
	O
Subsequent	O
3D	O
classification	O
using	O
a	O
2D	O
mask	O
comprising	O
PKI	O
and	O
domain	O
IV	O
of	O
eEF2	O
yielded	O
5	O
'	O
purified	O
'	O
classes	O
representing	O
Structures	O
I	O
through	O
V	O
.	O
Sub	O
-	O
classification	O
of	O
each	O
class	O
did	O
not	O
yield	O
additional	O
classes	O
,	O
but	O
helped	O
improve	O
density	O
in	O
the	O
PKI	O
region	O
of	O
class	O
III	O
(	O
estimated	O
resolution	O
and	O
percentage	O
of	O
particles	O
in	O
the	O
sub	O
-	O
classified	O
reconstruction	O
are	O
shown	O
in	O
parentheses	O
).	O
	O
Cryo	O
-	O
EM	O
structures	O
of	O
the	O
80S	O
•	O
TSV	O
IRES	O
bound	O
with	O
eEF2	O
•	O
GDP	O
•	O
sordarin	O
.	O
	O
(	O
a	O
)	O
Structures	O
I	O
through	O
V	O
.	O
In	O
all	O
panels	O
,	O
the	O
large	O
ribosomal	O
subunit	O
is	O
shown	O
in	O
cyan	O
;	O
the	O
small	O
subunit	O
in	O
light	O
yellow	O
(	O
head	O
)	O
and	O
wheat	O
-	O
yellow	O
(	O
body	O
);	O
the	O
TSV	O
IRES	O
in	O
red	O
,	O
eEF2	O
in	O
green	O
.	O
	O
We	O
sought	O
to	O
address	O
the	O
following	O
questions	O
by	O
structural	O
visualization	O
of	O
80S	O
•	O
IRES	O
•	O
eEF2	O
translocation	O
complexes	O
:	O
(	O
1	O
)	O
How	O
does	O
a	O
large	O
IRES	O
RNA	O
move	O
through	O
the	O
restricted	O
intersubunit	O
space	O
,	O
bringing	O
PKI	O
from	O
the	O
A	O
to	O
P	O
site	O
of	O
the	O
small	O
subunit	O
?	O
(	O
2	O
)	O
How	O
does	O
eEF2	O
mediate	O
IRES	O
translocation	O
?	O
(	O
3	O
)	O
Does	O
IRES	O
translocation	O
involve	O
large	O
rearrangements	O
in	O
the	O
ribosome	O
,	O
similar	O
to	O
tRNA	O
translocation	O
?	O
(	O
4	O
)	O
What	O
,	O
if	O
any	O
,	O
is	O
the	O
mechanistic	O
role	O
of	O
40S	O
head	O
rotation	O
in	O
IRES	O
translocation	O
?	O
	O
Maximum	O
-	O
likelihood	O
classification	O
using	O
FREALIGN	O
identified	O
five	O
IRES	O
-	O
eEF2	O
-	O
bound	O
ribosome	O
structures	O
within	O
a	O
single	O
sample	O
(	O
Figures	O
1	O
and	O
2	O
).	O
	O
The	O
structures	O
differ	O
in	O
the	O
positions	O
and	O
conformations	O
of	O
ribosomal	O
subunits	O
(	O
Figures	O
1b	O
and	O
2	O
),	O
IRES	O
RNA	O
(	O
Figures	O
3	O
and	O
4	O
)	O
and	O
eEF2	O
(	O
Figures	O
5	O
and	O
6	O
).	O
	O
18S	O
ribosomal	O
RNA	O
is	O
shown	O
and	O
ribosomal	O
proteins	O
are	O
omitted	O
for	O
clarity	O
.	O
	O
The	O
superpositions	O
of	O
structures	O
were	O
performed	O
by	O
structural	O
alignments	O
of	O
the	O
18S	O
ribosomal	O
RNAs	O
excluding	O
the	O
head	O
region	O
(	O
nt	O
1150	O
–	O
1620	O
).	O
	O
Structure	O
IV	O
adopts	O
a	O
slightly	O
rotated	O
conformation	O
(~	O
1	O
°).	O
	O
40S	O
head	O
swivel	O
	O
Comparison	O
of	O
the	O
TSV	O
IRES	O
and	O
eEF2	O
positions	O
in	O
Structures	O
I	O
through	O
V	O
.	O
	O
In	O
all	O
panels	O
,	O
superpositions	O
were	O
obtained	O
by	O
structural	O
alignments	O
of	O
the	O
18S	O
rRNAs	O
.	O
	O
Ribosomal	O
proteins	O
of	O
the	O
initiation	O
state	O
are	O
shown	O
in	O
gray	O
for	O
comparison	O
.	O
	O
Loop	O
1	O
.	O
1	O
and	O
stem	O
loops	O
4	O
and	O
5	O
of	O
the	O
IRES	O
are	O
labeled	O
.	O
	O
Positions	O
of	O
tRNAs	O
and	O
the	O
TSV	O
IRES	O
relative	O
to	O
the	O
A	O
-	O
site	O
finger	O
(	O
blue	O
,	O
nt	O
1008	O
–	O
1043	O
of	O
25S	O
rRNA	O
)	O
and	O
the	O
P	O
site	O
of	O
the	O
large	O
subunit	O
,	O
comprising	O
helix	O
84	O
of	O
25S	O
rRNA	O
(	O
nt	O
.	O
	O
Structures	O
of	O
80S	O
•	O
IRES	O
complexes	O
in	O
the	O
absence	O
of	O
eEF2	O
(	O
INIT	O
;	O
PDB	O
3J6Y	O
,)	O
and	O
in	O
the	O
presence	O
of	O
eEF2	O
(	O
this	O
work	O
)	O
are	O
shown	O
in	O
the	O
lower	O
row	O
and	O
labeled	O
.	O
	O
Interactions	O
of	O
the	O
TSV	O
IRES	O
with	O
uL5	O
and	O
eL42	O
.	O
	O
Pseudoknots	O
and	O
stem	O
loops	O
are	O
labeled	O
and	O
colored	O
as	O
in	O
(	O
a	O
).	O
	O
The	O
L1	O
.	O
1	O
region	O
remains	O
in	O
contact	O
with	O
the	O
L1	O
stalk	O
(	O
Figure	O
3	O
—	O
figure	O
supplement	O
3	O
).	O
	O
As	O
such	O
,	O
the	O
transition	O
from	O
the	O
initiation	O
state	O
to	O
Structure	O
I	O
involves	O
repositioning	O
of	O
SL3	O
around	O
the	O
A	O
-	O
site	O
finger	O
,	O
resembling	O
the	O
transition	O
between	O
the	O
pre	O
-	O
translocation	O
A	O
/	O
P	O
and	O
A	O
/	O
P	O
*	O
tRNA	O
.	O
	O
Another	O
local	O
rearrangement	O
concerns	O
loop	O
3	O
,	O
also	O
known	O
as	O
the	O
variable	O
loop	O
region	O
,	O
which	O
connects	O
the	O
ASL	O
-	O
and	O
mRNA	O
-	O
like	O
parts	O
of	O
PKI	O
.	O
	O
The	O
interaction	O
of	O
loop	O
3	O
backbone	O
with	O
uS7	O
resembles	O
that	O
of	O
the	O
anticodon	O
-	O
stem	O
loop	O
of	O
E	O
-	O
site	O
tRNA	O
in	O
the	O
post	O
-	O
translocation	O
80S	O
•	O
2tRNA	O
•	O
mRNA	O
structure	O
(	O
Figure	O
3	O
—	O
figure	O
supplement	O
5	O
).	O
	O
Ordering	O
of	O
loop	O
3	O
suggests	O
that	O
this	O
flexible	O
region	O
contributes	O
to	O
the	O
stabilization	O
of	O
the	O
PKI	O
domain	O
in	O
the	O
post	O
-	O
translocation	O
state	O
.	O
	O
(	O
c	O
)	O
Comparison	O
of	O
conformations	O
of	O
eEF2	O
•	O
sordarin	O
in	O
Structure	O
I	O
(	O
light	O
green	O
)	O
with	O
those	O
of	O
free	O
apo	O
-	O
eEF2	O
(	O
magenta	O
)	O
and	O
eEF2	O
•	O
sordarin	O
(	O
teal	O
).	O
	O
Superposition	O
was	O
obtained	O
by	O
structural	O
alignment	O
of	O
the	O
25S	O
rRNAs	O
.	O
	O
(	O
e	O
)	O
Comparison	O
of	O
the	O
GTP	O
-	O
like	O
conformation	O
of	O
eEF2	O
•	O
GDP	O
in	O
Structure	O
I	O
(	O
light	O
green	O
)	O
with	O
those	O
of	O
70S	O
-	O
bound	O
elongation	O
factors	O
EF	O
-	O
Tu	O
•	O
GDPCP	O
(	O
teal	O
)	O
and	O
EF	O
-	O
G	O
•	O
GDP	O
•	O
fusidic	O
acid	O
(	O
magenta	O
;	O
fusidic	O
acid	O
not	O
shown	O
).	O
(	O
f	O
)	O
Cryo	O
-	O
EM	O
density	O
showing	O
guanosine	O
diphosphate	O
bound	O
in	O
the	O
GTPase	O
center	O
(	O
green	O
)	O
next	O
to	O
the	O
sarcin	O
-	O
ricin	O
loop	O
of	O
25S	O
rRNA	O
(	O
cyan	O
)	O
of	O
Structure	O
II	O
.	O
(	O
g	O
)	O
Comparison	O
of	O
the	O
sordarin	O
-	O
binding	O
sites	O
in	O
the	O
ribosome	O
-	O
bound	O
(	O
light	O
green	O
;	O
Structure	O
II	O
)	O
and	O
isolated	O
eEF2	O
(	O
teal	O
).	O
	O
The	O
sarcin	O
-	O
ricin	O
loop	O
interacts	O
with	O
the	O
GTP	O
-	O
binding	O
site	O
of	O
eEF2	O
(	O
Figures	O
5d	O
and	O
f	O
).	O
	O
(	O
a	O
)	O
eEF2	O
(	O
green	O
)	O
interacts	O
only	O
with	O
the	O
body	O
in	O
Structure	O
I	O
(	O
eEF2	O
domains	O
are	O
labeled	O
with	O
roman	O
numerals	O
in	O
white	O
),	O
and	O
with	O
both	O
the	O
head	O
and	O
body	O
in	O
Structures	O
II	O
through	O
V	O
.	O
Colors	O
are	O
as	O
in	O
Figure	O
1	O
,	O
except	O
for	O
the	O
40S	O
structural	O
elements	O
that	O
contact	O
eEF2	O
,	O
which	O
are	O
labeled	O
and	O
shown	O
in	O
purple	O
.	O
(	O
b	O
)	O
Entry	O
of	O
eEF2	O
into	O
the	O
40S	O
A	O
site	O
,	O
from	O
Structure	O
I	O
through	O
V	O
.	O
Distances	O
to	O
the	O
A	O
-	O
site	O
accommodated	O
eEF2	O
(	O
Structure	O
V	O
)	O
are	O
shown	O
.	O
	O
Because	O
eEF2	O
is	O
rigidly	O
attached	O
to	O
the	O
60S	O
subunit	O
and	O
does	O
not	O
undergo	O
large	O
inter	O
-	O
subunit	O
rearrangements	O
,	O
gradual	O
entry	O
of	O
domain	O
IV	O
into	O
the	O
A	O
site	O
between	O
Structures	O
I	O
and	O
V	O
is	O
due	O
to	O
40S	O
subunit	O
rotation	O
and	O
head	O
swivel	O
.	O
	O
In	O
the	O
latter	O
,	O
PKI	O
is	O
stabilized	O
by	O
interactions	O
with	O
the	O
universally	O
conserved	O
decoding	O
-	O
center	O
nucleotides	O
G577	O
,	O
A1755	O
and	O
A1756	O
('	O
body	O
A	O
site	O
'),	O
as	O
in	O
the	O
A	O
-	O
site	O
tRNA	O
bound	O
complexes	O
.	O
	O
Domain	O
IV	O
is	O
partially	O
engaged	O
with	O
the	O
body	O
A	O
site	O
.	O
	O
The	O
trimethylamino	O
end	O
of	O
Diph699	O
packs	O
over	O
A1756	O
(	O
Figure	O
7	O
).	O
	O
In	O
translational	O
GTPases	O
,	O
switch	O
loops	O
I	O
and	O
II	O
are	O
involved	O
in	O
the	O
GTPase	O
activity	O
(	O
reviewed	O
in	O
).	O
	O
Next	O
to	O
GDP	O
,	O
the	O
C	O
-	O
terminal	O
part	O
of	O
the	O
switch	O
loop	O
(	O
aa	O
61	O
–	O
67	O
)	O
adopts	O
a	O
helical	O
fold	O
.	O
	O
The	O
decoding	O
center	O
residues	O
A1755	O
and	O
A1756	O
are	O
rearranged	O
to	O
pack	O
inside	O
helix	O
44	O
,	O
making	O
room	O
for	O
eEF2	O
.	O
	O
This	O
conformation	O
of	O
decoding	O
center	O
residues	O
is	O
also	O
observed	O
in	O
the	O
absence	O
of	O
A	O
-	O
site	O
ligands	O
.	O
	O
Structure	O
III	O
represents	O
a	O
highly	O
bent	O
IRES	O
with	O
PKI	O
captured	O
between	O
the	O
head	O
A	O
and	O
P	O
sites	O
	O
Among	O
the	O
five	O
structures	O
,	O
the	O
PKI	O
domain	O
is	O
least	O
ordered	O
in	O
Structure	O
III	O
and	O
lacks	O
density	O
for	O
SL3	O
.	O
	O
Unwinding	O
of	O
the	O
40S	O
head	O
also	O
positions	O
the	O
head	O
A	O
site	O
closer	O
to	O
the	O
body	O
A	O
site	O
.	O
	O
Four	O
views	O
(	O
scenes	O
)	O
are	O
shown	O
:	O
(	O
1	O
)	O
A	O
view	O
down	O
the	O
intersubunit	O
space	O
,	O
with	O
the	O
head	O
of	O
the	O
40S	O
subunit	O
oriented	O
toward	O
a	O
viewer	O
,	O
as	O
in	O
Figure	O
1a	O
;	O
(	O
2	O
)	O
A	O
view	O
at	O
the	O
solvent	O
side	O
of	O
the	O
40S	O
subunit	O
,	O
with	O
the	O
40S	O
head	O
shown	O
at	O
the	O
top	O
,	O
as	O
in	O
Figure	O
2	O
—	O
figure	O
supplement	O
1	O
;	O
(	O
3	O
)	O
A	O
view	O
down	O
at	O
the	O
subunit	O
interface	O
of	O
the	O
40S	O
subunit	O
;	O
(	O
4	O
)	O
A	O
close	O
-	O
up	O
view	O
of	O
the	O
decoding	O
center	O
(	O
A	O
site	O
)	O
and	O
the	O
P	O
site	O
,	O
as	O
in	O
Figure	O
2g	O
.	O
Each	O
scene	O
is	O
shown	O
twice	O
.	O
	O
Our	O
structures	O
reveal	O
previously	O
unseen	O
intermediate	O
states	O
of	O
eEF2	O
or	O
EF	O
-	O
G	O
engagement	O
with	O
the	O
A	O
site	O
,	O
providing	O
the	O
structural	O
basis	O
for	O
the	O
mechanism	O
of	O
translocase	O
action	O
.	O
	O
In	O
the	O
first	O
sub	O
-	O
step	O
(	O
Structures	O
I	O
to	O
IV	O
),	O
the	O
hind	O
end	O
advances	O
from	O
the	O
A	O
to	O
the	O
P	O
site	O
and	O
approaches	O
the	O
front	O
end	O
,	O
which	O
remains	O
attached	O
to	O
the	O
40S	O
surface	O
.	O
	O
Upon	O
translocation	O
,	O
the	O
GCU	O
start	O
codon	O
is	O
positioned	O
in	O
the	O
A	O
site	O
(	O
Structure	O
V	O
),	O
ready	O
for	O
interaction	O
with	O
Ala	O
-	O
tRNAAla	O
upon	O
eEF2	O
departure	O
.	O
	O
Recent	O
studies	O
have	O
shown	O
that	O
in	O
some	O
cases	O
a	O
fraction	O
of	O
IGR	O
IRES	O
-	O
driven	O
translation	O
results	O
from	O
an	O
alternative	O
reading	O
frame	O
,	O
which	O
is	O
shifted	O
by	O
one	O
nucleotide	O
relative	O
to	O
the	O
normal	O
ORF	O
.	O
	O
In	O
our	O
structures	O
,	O
the	O
IRES	O
presents	O
to	O
the	O
decoding	O
center	O
a	O
pre	O
-	O
translocated	O
or	O
fully	O
translocated	O
ORF	O
,	O
rather	O
than	O
a	O
+	O
1	O
(	O
more	O
translocated	O
)	O
ORF	O
,	O
suggesting	O
that	O
eEF2	O
does	O
not	O
induce	O
a	O
highly	O
populated	O
fraction	O
of	O
+	O
1	O
shifted	O
IRES	O
mRNAs	O
.	O
	O
This	O
is	O
consistent	O
with	O
the	O
observations	O
that	O
the	O
intergenic	O
IRESs	O
are	O
prone	O
to	O
reverse	O
translocation	O
.	O
	O
In	O
the	O
initiation	O
state	O
,	O
the	O
IRES	O
resembles	O
a	O
pre	O
-	O
translocation	O
2tRNA	O
•	O
mRNA	O
complex	O
reduced	O
to	O
the	O
A	O
/	O
P	O
-	O
tRNA	O
anticodon	O
-	O
stem	O
loop	O
and	O
elbow	O
in	O
the	O
A	O
site	O
and	O
the	O
P	O
/	O
E	O
-	O
tRNA	O
elbow	O
contacting	O
the	O
L1	O
stalk	O
.	O
	O
Because	O
the	O
anticodon	O
-	O
stem	O
loop	O
of	O
the	O
A	O
-	O
tRNA	O
is	O
sufficient	O
for	O
translocation	O
completion	O
,	O
we	O
ascribe	O
the	O
meta	O
-	O
stability	O
of	O
the	O
post	O
-	O
translocation	O
IRES	O
to	O
the	O
absence	O
of	O
the	O
P	O
/	O
E	O
-	O
tRNA	O
elements	O
,	O
either	O
the	O
ASL	O
or	O
the	O
acceptor	O
arm	O
,	O
or	O
both	O
.	O
	O
Translocases	O
are	O
efficient	O
enzymes	O
.	O
	O
EF	O
-	O
G	O
enhances	O
the	O
translocation	O
rate	O
by	O
several	O
orders	O
of	O
magnitude	O
,	O
aided	O
by	O
an	O
additional	O
2	O
-	O
to	O
50	O
-	O
fold	O
boost	O
from	O
GTP	O
hydrolysis	O
.	O
	O
Due	O
to	O
the	O
lack	O
of	O
structures	O
of	O
translocation	O
intermediates	O
,	O
the	O
mechanistic	O
role	O
of	O
eEF2	O
/	O
EF	O
-	O
G	O
is	O
not	O
fully	O
understood	O
.	O
	O
The	O
unlocking	O
model	O
of	O
the	O
ribosome	O
•	O
2tRNA	O
•	O
mRNA	O
pre	O
-	O
translocation	O
complex	O
has	O
been	O
proposed	O
decades	O
ago	O
and	O
functional	O
requirement	O
of	O
the	O
translocase	O
in	O
this	O
process	O
has	O
been	O
implicated	O
.	O
	O
This	O
destabilization	O
allows	O
PKI	O
to	O
detach	O
from	O
the	O
body	O
A	O
site	O
upon	O
spontaneous	O
reverse	O
40S	O
body	O
rotation	O
,	O
while	O
maintaining	O
interactions	O
with	O
the	O
head	O
A	O
site	O
.	O
	O
In	O
the	O
fully	O
-	O
rotated	O
pre	O
-	O
translocation	O
-	O
like	O
Structure	O
I	O
,	O
an	O
additional	O
interaction	O
exists	O
.	O
	O
We	O
propose	O
that	O
the	O
shift	O
of	O
domain	O
III	O
by	O
uS12	O
initiates	O
intra	O
-	O
domain	O
rearrangements	O
in	O
eEF2	O
,	O
which	O
unstack	O
the	O
β	O
-	O
platform	O
of	O
domain	O
III	O
from	O
that	O
of	O
domain	O
V	O
.	O
This	O
would	O
result	O
in	O
a	O
conformation	O
characteristic	O
of	O
free	O
eEF2	O
and	O
EF	O
-	O
G	O
in	O
which	O
the	O
β	O
-	O
platforms	O
are	O
nearly	O
perpendicular	O
.	O
	O
Sordarin	O
is	O
a	O
potent	O
antifungal	O
antibiotic	O
that	O
inhibits	O
translation	O
.	O
	O
Although	O
our	O
complex	O
was	O
assembled	O
using	O
eEF2	O
•	O
GTP	O
,	O
density	O
maps	O
clearly	O
show	O
GDP	O
and	O
Mg2	O
+	O
in	O
each	O
structure	O
(	O
Figure	O
5g	O
).	O
	O
In	O
all	O
five	O
structures	O
,	O
sordarin	O
is	O
bound	O
between	O
domains	O
III	O
and	O
V	O
of	O
eEF2	O
,	O
stabilized	O
by	O
hydrophobic	O
interactions	O
identical	O
to	O
those	O
in	O
the	O
isolated	O
eEF2	O
•	O
sordarin	O
complex	O
(	O
Figures	O
5g	O
and	O
h	O
).	O
	O
Implications	O
for	O
tRNA	O
and	O
mRNA	O
translocation	O
during	O
translation	O
	O
First	O
,	O
we	O
propose	O
that	O
tRNA	O
and	O
IRES	O
translocations	O
occur	O
via	O
the	O
same	O
general	O
trajectory	O
.	O
	O
This	O
is	O
consistent	O
with	O
the	O
idea	O
of	O
a	O
rather	O
flat	O
energy	O
landscape	O
of	O
translocation	O
,	O
suggested	O
by	O
recent	O
work	O
that	O
measured	O
mechanical	O
work	O
produced	O
by	O
the	O
ribosome	O
during	O
translocation	O
.	O
	O
We	O
note	O
that	O
four	O
of	O
our	O
near	O
-	O
atomic	O
resolution	O
maps	O
comprised	O
~	O
30	O
,	O
000	O
particles	O
each	O
,	O
the	O
minimum	O
number	O
required	O
for	O
a	O
near	O
-	O
atomic	O
-	O
resolution	O
reconstruction	O
of	O
the	O
ribosome	O
.	O
	O
This	O
difference	O
likely	O
accounts	O
for	O
the	O
inefficient	O
translocation	O
of	O
the	O
IRES	O
,	O
which	O
is	O
difficult	O
to	O
stabilize	O
in	O
the	O
post	O
-	O
translocation	O
state	O
and	O
therefore	O
is	O
prone	O
to	O
reverse	O
translocation	O
.	O
	O
The	O
uniformity	O
of	O
ribosome	O
dynamics	O
underscores	O
the	O
idea	O
that	O
translocation	O
is	O
an	O
inherent	O
and	O
structurally	O
-	O
optimized	O
property	O
of	O
the	O
ribosome	O
,	O
supported	O
also	O
by	O
translocation	O
activity	O
in	O
the	O
absence	O
of	O
the	O
elongation	O
factor	O
.	O
	O
Our	O
current	O
understanding	O
of	O
macromolecular	O
machines	O
,	O
such	O
as	O
the	O
ribosome	O
,	O
is	O
often	O
limited	O
by	O
a	O
gap	O
between	O
biophysical	O
/	O
biochemical	O
studies	O
and	O
structural	O
studies	O
.	O
	O
For	O
example	O
,	O
Förster	O
resonance	O
energy	O
transfer	O
can	O
provide	O
insight	O
into	O
the	O
macromolecular	O
dynamics	O
of	O
an	O
assembly	O
at	O
the	O
single	O
-	O
molecule	O
level	O
but	O
is	O
limited	O
to	O
specifically	O
labeled	O
locations	O
within	O
the	O
assembly	O
.	O
	O
The	O
classification	O
,	O
which	O
followed	O
an	O
initial	O
alignment	O
of	O
all	O
particles	O
to	O
a	O
single	O
reference	O
,	O
required	O
about	O
130	O
,	O
000	O
CPU	O
hours	O
or	O
about	O
five	O
to	O
six	O
full	O
days	O
on	O
a	O
1000	O
-	O
CPU	O
cluster	O
.	O
	O
The	O
N	O
-	O
terminal	O
propeptides	O
protecting	O
the	O
active	O
-	O
site	O
threonines	O
are	O
autocatalytically	O
released	O
only	O
on	O
completion	O
of	O
assembly	O
.	O
	O
This	O
mechanism	O
,	O
however	O
,	O
cannot	O
explain	O
autocatalytic	O
precursor	O
processing	O
because	O
in	O
the	O
immature	O
active	O
sites	O
,	O
Thr1N	O
is	O
part	O
of	O
the	O
peptide	O
bond	O
with	O
Gly	O
(-	O
1	O
),	O
the	O
bond	O
that	O
needs	O
to	O
be	O
hydrolysed	O
.	O
	O
Inactivation	O
of	O
proteasome	O
subunits	O
by	O
T1A	B-mutant
mutations	O
	O
Sequencing	O
of	O
the	O
plasmids	O
,	O
testing	O
them	O
in	O
both	O
published	O
yeast	O
strain	O
backgrounds	O
and	O
site	O
-	O
directed	O
mutagenesis	O
revealed	O
that	O
the	O
β5	B-mutant
-	I-mutant
T1A	I-mutant
mutant	O
pp	O
cis	O
is	O
viable	O
,	O
but	O
suffers	O
from	O
a	O
marked	O
growth	O
defect	O
that	O
requires	O
extended	O
incubation	O
of	O
4	O
–	O
5	O
days	O
for	O
initial	O
colony	O
formation	O
(	O
Table	O
1	O
and	O
Supplementary	O
Methods	O
).	O
	O
For	O
subunit	O
β1	O
,	O
this	O
process	O
was	O
previously	O
inferred	O
to	O
require	O
that	O
the	O
propeptide	O
residue	O
at	O
position	O
(-	O
2	O
)	O
of	O
the	O
subunit	O
precursor	O
occupies	O
the	O
S1	O
specificity	O
pocket	O
of	O
the	O
substrate	O
-	O
binding	O
channel	O
formed	O
by	O
amino	O
acid	O
45	O
(	O
for	O
details	O
see	O
Supplementary	O
Note	O
2	O
).	O
	O
Here	O
we	O
again	O
analysed	O
the	O
β1	B-mutant
-	I-mutant
T1A	I-mutant
mutant	O
crystallographically	O
but	O
in	O
addition	O
determined	O
the	O
structures	O
of	O
the	O
β2	B-mutant
-	I-mutant
T1A	I-mutant
single	O
and	O
β1	B-mutant
-	I-mutant
T1A	I-mutant
-	I-mutant
β2	I-mutant
-	I-mutant
T1A	I-mutant
double	O
mutants	O
(	O
Protein	O
Data	O
Bank	O
(	O
PDB	O
)	O
entry	O
codes	O
are	O
provided	O
in	O
Supplementary	O
Table	O
1	O
).	O
	O
Instead	O
,	O
the	O
plasticity	O
of	O
the	O
β5	O
S1	O
pocket	O
caused	O
by	O
the	O
rotational	O
flexibility	O
of	O
Met45	O
might	O
prevent	O
stable	O
accommodation	O
of	O
His	O
(-	O
2	O
)	O
in	O
the	O
S1	O
site	O
and	O
thus	O
also	O
promote	O
its	O
immediate	O
release	O
after	O
autolysis	O
.	O
	O
Structural	O
analyses	O
revealed	O
that	O
the	O
propeptides	O
of	O
all	O
mutant	O
yCPs	O
shared	O
residual	O
2FO	O
–	O
FC	O
electron	O
densities	O
.	O
	O
By	O
contrast	O
,	O
the	O
prosegments	O
of	O
the	O
β5	B-mutant
-	I-mutant
H	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
L	I-mutant
-	I-mutant
T1A	I-mutant
and	O
the	O
β5	B-mutant
-	I-mutant
H	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
T	I-mutant
-	I-mutant
T1A	I-mutant
mutants	O
were	O
significantly	O
better	O
resolved	O
in	O
the	O
2FO	O
–	O
FC	O
electron	O
-	O
density	O
maps	O
yet	O
not	O
at	O
full	O
occupancy	O
(	O
Supplementary	O
Fig	O
.	O
4b	O
,	O
c	O
and	O
Supplementary	O
Table	O
1	O
),	O
suggesting	O
that	O
the	O
natural	O
propeptide	O
bearing	O
His	O
(-	O
2	O
)	O
is	O
most	O
favourable	O
.	O
	O
This	O
result	O
proves	O
that	O
the	O
naturally	O
occurring	O
His	O
(-	O
2	O
)	O
of	O
the	O
β5	O
propeptide	O
does	O
not	O
stably	O
fit	O
into	O
the	O
S1	O
site	O
.	O
	O
Bearing	O
in	O
mind	O
that	O
in	O
contrast	O
to	O
Thr	O
(-	O
2	O
)	O
in	O
β2	O
,	O
Leu	O
(-	O
2	O
)	O
in	O
subunit	O
β1	O
is	O
not	O
conserved	O
among	O
species	O
(	O
Supplementary	O
Fig	O
.	O
3a	O
),	O
we	O
created	O
a	O
β2	B-mutant
-	I-mutant
T	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
V	I-mutant
proteasome	O
mutant	O
.	O
	O
However	O
,	O
in	O
the	O
immature	O
particle	O
Thr1NH2	O
is	O
blocked	O
by	O
the	O
propeptide	O
and	O
cannot	O
activate	O
Thr1Oγ	O
.	O
	O
Instead	O
,	O
Lys33NH2	O
,	O
which	O
is	O
in	O
hydrogen	O
-	O
bonding	O
distance	O
to	O
Thr1Oγ	O
(	O
2	O
.	O
7	O
Å	O
)	O
in	O
all	O
catalytically	O
active	O
β	O
subunits	O
(	O
Fig	O
.	O
3a	O
,	O
b	O
),	O
was	O
proposed	O
to	O
serve	O
as	O
the	O
proton	O
acceptor	O
.	O
	O
This	O
water	O
hydrogen	O
bonds	O
also	O
to	O
Arg19O	O
(∼	O
3	O
.	O
0	O
Å	O
)	O
and	O
Asp17Oδ	O
(∼	O
3	O
.	O
0	O
Å	O
),	O
and	O
thereby	O
presumably	O
enables	O
residual	O
activity	O
of	O
the	O
mutant	O
.	O
	O
The	O
ChT	O
-	O
L	O
activity	O
of	O
the	O
β5	B-mutant
-	I-mutant
D17N	I-mutant
pp	O
in	O
trans	O
CP	O
towards	O
the	O
canonical	O
β5	O
model	O
substrates	O
N	O
-	O
succinyl	O
-	O
Leu	O
-	O
Leu	O
-	O
Val	O
-	O
Tyr	O
-	O
7	O
-	O
amino	O
-	O
4	O
-	O
methylcoumarin	O
(	O
Suc	O
-	O
LLVY	O
-	O
AMC	O
)	O
and	O
carboxybenzyl	O
-	O
Gly	O
-	O
Gly	O
-	O
Leu	O
-	O
para	O
-	O
nitroanilide	O
(	O
Z	O
-	O
GGL	O
-	O
pNA	O
)	O
was	O
severely	O
reduced	O
(	O
Supplementary	O
Fig	O
.	O
6b	O
),	O
confirming	O
that	O
Asp17	O
is	O
of	O
fundamental	O
importance	O
for	O
the	O
catalytic	O
activity	O
of	O
the	O
mature	O
proteasome	O
.	O
	O
Strikingly	O
,	O
although	O
the	O
X	O
-	O
ray	O
data	O
on	O
the	O
β5	B-mutant
-	I-mutant
D17N	I-mutant
mutant	O
with	O
the	O
propeptide	O
expressed	O
in	O
cis	O
and	O
in	O
trans	O
looked	O
similar	O
,	O
there	O
was	O
a	O
pronounced	O
difference	O
in	O
their	O
growth	O
phenotypes	O
observed	O
(	O
Supplementary	O
Fig	O
.	O
6a	O
and	O
Supplementary	O
Fig	O
.	O
7b	O
).	O
	O
The	O
β5	B-mutant
-	I-mutant
D166N	I-mutant
pp	O
cis	O
yeast	O
mutant	O
is	O
significantly	O
impaired	O
in	O
growth	O
and	O
its	O
ChT	O
-	O
L	O
activity	O
is	O
drastically	O
reduced	O
(	O
Supplementary	O
Fig	O
.	O
6a	O
,	O
b	O
and	O
Table	O
1	O
).	O
	O
The	O
hydrogen	O
bonds	O
involving	O
Ser169OH	O
are	O
intact	O
and	O
may	O
account	O
for	O
residual	O
substrate	O
turnover	O
.	O
	O
Together	O
,	O
these	O
observations	O
suggest	O
that	O
efficient	O
peptide	O
-	O
bond	O
hydrolysis	O
requires	O
that	O
Lys33NH2	O
hydrogen	O
bonds	O
to	O
the	O
active	O
site	O
nucleophile	O
.	O
	O
Activity	O
assays	O
with	O
the	O
β5	O
-	O
specific	O
substrate	O
Suc	O
-	O
LLVY	O
-	O
AMC	O
demonstrated	O
that	O
the	O
ChT	O
-	O
L	O
activity	O
of	O
the	O
T1S	B-mutant
mutant	O
is	O
reduced	O
by	O
40	O
–	O
45	O
%	O
compared	O
with	O
WT	O
proteasomes	O
depending	O
on	O
the	O
incubation	O
temperature	O
(	O
Fig	O
.	O
4b	O
and	O
Supplementary	O
Fig	O
.	O
9c	O
).	O
	O
Compared	O
with	O
Thr1Oγ	O
in	O
WT	O
CP	O
structures	O
,	O
Ser1Oγ	O
is	O
rotated	O
by	O
60	O
°.	O
	O
In	O
addition	O
,	O
they	O
prevent	O
irreversible	O
inactivation	O
of	O
the	O
Thr1	O
N	O
terminus	O
by	O
N	O
-	O
acetylation	O
.	O
	O
However	O
,	O
removal	O
of	O
the	O
β5	O
prosegment	O
or	O
any	O
interference	O
with	O
its	O
cleavage	O
causes	O
severe	O
phenotypic	O
defects	O
.	O
	O
On	O
the	O
basis	O
of	O
the	O
numerous	O
CP	O
:	O
ligand	O
complexes	O
solved	O
during	O
the	O
past	O
18	O
years	O
and	O
in	O
the	O
current	O
study	O
,	O
we	O
provide	O
a	O
revised	O
interpretation	O
of	O
proteasome	O
active	O
-	O
site	O
architecture	O
.	O
	O
We	O
propose	O
a	O
catalytic	O
triad	O
for	O
the	O
active	O
site	O
of	O
the	O
CP	O
consisting	O
of	O
residues	O
Thr1	O
,	O
Lys33	O
and	O
Asp	O
/	O
Glu17	O
,	O
which	O
are	O
conserved	O
among	O
all	O
proteolytically	O
active	O
eukaryotic	O
,	O
bacterial	O
and	O
archaeal	O
proteasome	O
subunits	O
.	O
	O
Cleavage	O
of	O
the	O
scissile	O
peptide	O
bond	O
requires	O
protonation	O
of	O
the	O
emerging	O
free	O
amine	O
,	O
and	O
in	O
the	O
proteasome	O
,	O
the	O
Thr1	O
amine	O
group	O
is	O
likely	O
to	O
assume	O
this	O
function	O
.	O
	O
Analogously	O
,	O
Thr1NH3	O
+	O
might	O
promote	O
the	O
bivalent	O
reaction	O
mode	O
of	O
epoxyketone	O
inhibitors	O
by	O
protonating	O
the	O
epoxide	O
moiety	O
to	O
create	O
a	O
positively	O
charged	O
trivalent	O
oxygen	O
atom	O
that	O
is	O
subsequently	O
nucleophilically	O
attacked	O
by	O
Thr1NH2	O
.	O
	O
The	O
residues	O
Ser129	O
and	O
Asp166	O
are	O
expected	O
to	O
increase	O
the	O
pKa	O
value	O
of	O
Thr1N	O
,	O
thereby	O
favouring	O
its	O
charged	O
state	O
.	O
	O
Consistent	O
with	O
playing	O
an	O
essential	O
role	O
in	O
proton	O
shuttling	O
,	O
the	O
mutation	O
D166A	B-mutant
prevents	O
autolysis	O
of	O
the	O
archaeal	O
CP	O
and	O
the	O
exchange	O
D166N	B-mutant
impairs	O
catalytic	O
activity	O
of	O
the	O
yeast	O
CP	O
about	O
60	O
%.	O
	O
While	O
Lys33NH2	O
and	O
Asp17Oδ	O
are	O
required	O
to	O
deprotonate	O
the	O
Thr1	O
hydroxyl	O
side	O
chain	O
,	O
Ser129OH	O
and	O
Asp166OH	O
serve	O
to	O
protonate	O
the	O
N	O
-	O
terminal	O
amine	O
group	O
of	O
Thr1	O
.	O
	O
Structural	O
analyses	O
support	O
these	O
findings	O
with	O
the	O
T1S	B-mutant
mutant	O
and	O
provide	O
an	O
explanation	O
for	O
the	O
strict	O
use	O
of	O
Thr	O
residues	O
in	O
proteasomes	O
.	O
	O
Notably	O
,	O
proteolytically	O
active	O
proteasome	O
subunits	O
from	O
archaea	O
,	O
yeast	O
and	O
mammals	O
,	O
including	O
constitutive	O
,	O
immuno	O
-	O
and	O
thymoproteasome	O
subunits	O
,	O
either	O
encode	O
Thr	O
or	O
Ile	O
at	O
position	O
3	O
,	O
indicating	O
the	O
importance	O
of	O
the	O
Cγ	O
for	O
fixing	O
the	O
position	O
of	O
the	O
nucleophilic	O
Thr1	O
.	O
	O
The	O
major	O
determinant	O
of	O
the	O
S1	O
specificity	O
pocket	O
,	O
residue	O
45	O
,	O
is	O
depicted	O
.	O
	O
Note	O
the	O
tight	O
conformation	O
of	O
Gly	O
(-	O
1	O
)	O
and	O
Ala1	O
before	O
propeptide	O
removal	O
(	O
G	O
(-	O
1	O
)	O
turn	O
;	O
cyan	O
double	O
arrow	O
)	O
compared	O
with	O
the	O
relaxed	O
,	O
processed	O
WT	O
active	O
-	O
site	O
Thr1	O
(	O
red	O
double	O
arrow	O
).	O
	O
The	O
black	O
arrow	O
indicates	O
the	O
attack	O
of	O
Thr1Oγ	O
onto	O
the	O
carbonyl	O
carbon	O
atom	O
of	O
Gly	O
(-	O
1	O
).	O
	O
While	O
residue	O
(-	O
2	O
)	O
of	O
the	O
β1	O
and	O
β2	O
prosegments	O
fit	O
the	O
S1	O
pocket	O
,	O
His	O
(-	O
2	O
)	O
of	O
the	O
β5	O
propeptide	O
occupies	O
the	O
S2	O
pocket	O
.	O
	O
The	O
(-	O
2	O
)	O
residues	O
of	O
both	O
prosegments	O
point	O
into	O
the	O
S1	O
pocket	O
,	O
but	O
only	O
Thr	O
(-	O
2	O
)	O
OH	O
of	O
β2	O
forms	O
a	O
hydrogen	O
bridge	O
to	O
Gly	O
(-	O
1	O
)	O
O	O
(	O
black	O
dashed	O
line	O
).	O
	O
(	O
d	O
)	O
Structural	O
superposition	O
of	O
the	O
matured	O
β2	O
active	O
site	O
,	O
the	O
WT	O
β2	B-mutant
-	I-mutant
T1A	I-mutant
propeptide	O
and	O
the	O
β2	B-mutant
-	I-mutant
T	I-mutant
(-	I-mutant
2	I-mutant
)	I-mutant
V	I-mutant
mutant	O
propeptide	O
.	O
	O
The	O
Thr1	O
N	O
terminus	O
is	O
engaged	O
in	O
hydrogen	O
bonds	O
with	O
Ser129Oγ	O
,	O
the	O
carbonyl	O
oxygen	O
of	O
residue	O
168	O
,	O
Ser169Oγ	O
and	O
Asp166Oδ	O
.	O
(	O
b	O
)	O
The	O
orientations	O
of	O
the	O
active	O
-	O
site	O
residues	O
involved	O
in	O
hydrogen	O
bonding	O
are	O
strictly	O
conserved	O
in	O
each	O
proteolytic	O
centre	O
,	O
as	O
shown	O
by	O
superposition	O
of	O
the	O
β	O
subunits	O
.	O
	O
In	O
the	O
latter	O
,	O
a	O
water	O
molecule	O
(	O
red	O
sphere	O
)	O
is	O
found	O
at	O
the	O
position	O
where	O
in	O
the	O
WT	O
structure	O
the	O
side	O
chain	O
amine	O
group	O
of	O
Lys33	O
is	O
located	O
.	O
	O
Note	O
,	O
the	O
strong	O
interaction	O
with	O
the	O
water	O
molecule	O
causes	O
a	O
minor	O
shift	O
of	O
Thr1	O
,	O
while	O
all	O
other	O
active	O
-	O
site	O
residues	O
remain	O
in	O
place	O
.	O
	O
The	O
charged	O
Thr1	O
N	O
terminus	O
may	O
engage	O
in	O
the	O
orientation	O
of	O
the	O
amide	O
moiety	O
and	O
donate	O
a	O
proton	O
to	O
the	O
emerging	O
N	O
terminus	O
of	O
the	O
C	O
-	O
terminal	O
cleavage	O
product	O
.	O
	O
The	O
2FO	O
–	O
FC	O
electron	O
-	O
density	O
maps	O
(	O
blue	O
mesh	O
)	O
for	O
Ser1	O
(	O
brown	O
)	O
and	O
the	O
covalently	O
bound	O
ligands	O
(	O
green	O
;	O
only	O
the	O
P1	O
site	O
(	O
Leu1	O
)	O
is	O
shown	O
)	O
are	O
contoured	O
at	O
1σ	O
.	O
	O
The	O
Taf14	O
YEATS	O
domain	O
is	O
a	O
reader	O
of	O
histone	O
crotonylation	O
	O
Owing	O
to	O
some	O
differences	O
in	O
their	O
genomic	O
distribution	O
,	O
the	O
crotonyllysine	O
and	O
acetyllysine	O
(	O
Kac	O
)	O
modifications	O
have	O
been	O
linked	O
to	O
distinct	O
functional	O
outcomes	O
.	O
	O
A	O
recent	O
survey	O
of	O
bromodomains	O
(	O
BDs	O
)	O
demonstrates	O
that	O
only	O
one	O
BD	O
associates	O
very	O
weakly	O
with	O
a	O
crotonylated	O
peptide	O
,	O
however	O
it	O
binds	O
more	O
tightly	O
to	O
acetylated	O
peptides	O
,	O
inferring	O
that	O
bromodomains	O
do	O
not	O
possess	O
physiologically	O
relevant	O
crotonyllysine	O
binding	O
activity	O
.	O
	O
The	O
most	O
striking	O
feature	O
of	O
the	O
crotonyllysine	O
recognition	O
mechanism	O
is	O
the	O
unique	O
coordination	O
of	O
crotonylated	O
lysine	O
residue	O
.	O
	O
The	O
π	O
bond	O
conjugation	O
of	O
the	O
crotonyl	O
group	O
gives	O
rise	O
to	O
a	O
dipole	O
moment	O
of	O
the	O
alkene	O
moiety	O
,	O
resulting	O
in	O
a	O
partial	O
positive	O
charge	O
on	O
the	O
β	O
-	O
carbon	O
(	O
Cβ	O
)	O
and	O
a	O
partial	O
negative	O
charge	O
on	O
the	O
α	O
-	O
carbon	O
(	O
Cα	O
).	O
	O
The	O
dissociation	O
constant	O
(	O
Kd	O
)	O
for	O
the	O
Taf14	O
YEATS	O
-	O
H3K9cr5	O
-	O
13	O
complex	O
was	O
found	O
to	O
be	O
9	O
.	O
5	O
μM	O
,	O
as	O
measured	O
by	O
fluorescence	O
spectroscopy	O
(	O
Supplementary	O
Fig	O
.	O
2c	O
).	O
	O
Towards	O
this	O
end	O
,	O
we	O
probed	O
extracts	O
derived	O
from	O
yeast	O
cells	O
in	O
which	O
major	O
yeast	O
HATs	O
(	O
HAT1	O
,	O
Gcn5	O
,	O
and	O
Rtt109	O
)	O
or	O
HDACs	O
(	O
Rpd3	O
,	O
Hos1	O
,	O
and	O
Hos2	O
)	O
were	O
deleted	O
.	O
	O
In	O
contrast	O
,	O
binding	O
of	O
H3K9ac	O
resulted	O
in	O
an	O
intermediate	O
exchange	O
,	O
which	O
is	O
characteristic	O
of	O
a	O
weaker	O
association	O
.	O
	O
The	O
preference	O
for	O
H3K9cr	O
over	O
H3K9ac	O
,	O
H3K9pr	O
and	O
H3K9bu	O
was	O
supported	O
by	O
1H	O
,	O
15N	O
HSQC	O
titration	O
experiments	O
.	O
	O
H3K9cr	O
is	O
a	O
selective	O
target	O
of	O
the	O
Taf14	O
YEATS	O
domain	O
	O
Cellular	O
homeostasis	O
requires	O
correct	O
delivery	O
of	O
cell	O
-	O
surface	O
receptor	O
proteins	O
(	O
cargo	O
)	O
to	O
their	O
target	O
subcellular	O
compartments	O
.	O
	O
The	O
adapter	O
proteins	O
Tom1	O
and	O
Tollip	O
are	O
involved	O
in	O
sorting	O
of	O
ubiquitinated	O
cargo	O
in	O
endosomal	O
compartments	O
.	O
	O
Recruitment	O
of	O
Tom1	O
to	O
the	O
endosomal	O
compartments	O
is	O
mediated	O
by	O
its	O
GAT	O
domain	O
’	O
s	O
association	O
to	O
Tollip	O
’	O
s	O
Tom1	O
-	O
binding	O
domain	O
(	O
TBD	O
).	O
	O
Subject	O
area	O
Biology	O
More	O
specific	O
subject	O
area	O
Structural	O
biology	O
Type	O
of	O
data	O
Table	O
,	O
text	O
file	O
,	O
graph	O
,	O
figures	O
How	O
data	O
was	O
acquired	O
Circular	O
dichroism	O
and	O
NMR	O
.	O
	O
Analysis	O
of	O
the	O
far	O
-	O
UV	O
circular	O
dichroism	O
(	O
CD	O
)	O
spectrum	O
of	O
the	O
Tom	O
1	O
GAT	O
domain	O
(	O
Fig	O
.	O
1	O
)	O
predicts	O
58	O
.	O
7	O
%	O
α	O
-	O
helix	O
,	O
3	O
%	O
β	O
-	O
strand	O
,	O
15	O
.	O
5	O
%	O
turn	O
,	O
and	O
22	O
.	O
8	O
%	O
disordered	O
regions	O
.	O
	O
Helices	O
are	O
shown	O
in	O
orange	O
,	O
whereas	O
loops	O
are	O
colored	O
in	O
green	O
.	O
(	O
B	O
)	O
Ribbon	O
illustration	O
of	O
the	O
Tom1	O
GAT	O
domain	O
.	O
	O
deviations	O
were	O
obtained	O
by	O
superimposing	O
residues	O
215	O
–	O
309	O
of	O
Tom1	O
GAT	O
among	O
10	O
lowest	O
energy	O
refined	O
structures	O
.	O
	O
PGRMC1	O
is	O
a	O
member	O
of	O
the	O
membrane	O
-	O
associated	O
progesterone	O
receptor	O
(	O
MAPR	O
)	O
family	O
with	O
a	O
cytochrome	O
b5	O
-	O
like	O
haem	O
-	O
binding	O
region	O
,	O
and	O
is	O
known	O
to	O
be	O
highly	O
expressed	O
in	O
various	O
types	O
of	O
cancers	O
.	O
	O
These	O
histidines	O
are	O
missing	O
in	O
PGRMC1	O
,	O
and	O
the	O
haem	O
iron	O
is	O
five	O
-	O
coordinated	O
by	O
Tyr113	O
(	O
Y113	O
)	O
alone	O
(	O
Fig	O
.	O
1b	O
and	O
Supplementary	O
Fig	O
.	O
3	O
).	O
	O
However	O
,	O
at	O
the	O
interfaces	O
of	O
the	O
other	O
possible	O
dimeric	O
structures	O
(	O
Supplementary	O
Fig	O
.	O
6a	O
,	O
chain	O
A	O
–	O
A	O
″;	O
cyan	O
and	O
chain	O
A	O
–	O
B	O
;	O
violet	O
),	O
no	O
significant	O
difference	O
was	O
observed	O
.	O
	O
It	O
should	O
be	O
noted	O
that	O
a	O
disulfide	O
bond	O
between	O
two	O
Cys129	O
residues	O
is	O
observed	O
in	O
the	O
crystal	O
of	O
PGRMC1	O
(	O
Fig	O
.	O
1a	O
),	O
while	O
Cys129	O
is	O
not	O
conserved	O
among	O
the	O
MAPR	O
family	O
proteins	O
(	O
Supplementary	O
Fig	O
.	O
5a	O
).	O
	O
The	O
current	O
analytical	O
data	O
confirmed	O
that	O
apo	O
-	O
PGRMC1	O
monomer	O
converts	O
into	O
dimer	O
by	O
binding	O
to	O
haem	O
in	O
solution	O
(	O
Table	O
2	O
).	O
	O
Furthermore	O
,	O
the	O
UV	O
-	O
visible	O
spectrum	O
of	O
the	O
wild	O
type	O
PGRMC1	O
was	O
the	O
same	O
as	O
that	O
of	O
the	O
C129S	B-mutant
mutant	O
of	O
PGRMC1	O
,	O
and	O
the	O
R	O
/	O
Z	O
ratio	O
determined	O
by	O
the	O
intensities	O
between	O
the	O
Soret	O
band	O
(	O
394	O
nm	O
)	O
peak	O
and	O
the	O
274	O
-	O
nm	O
peak	O
showed	O
that	O
these	O
proteins	O
were	O
fully	O
loaded	O
with	O
haem	O
(	O
Supplementary	O
Fig	O
.	O
12	O
).	O
	O
To	O
examine	O
the	O
inhibitory	O
effects	O
of	O
CO	O
on	O
haem	O
-	O
mediated	O
PGRMC1	O
dimerization	O
,	O
SV	O
-	O
AUC	O
analysis	O
was	O
carried	O
out	O
.	O
	O
By	O
binding	O
with	O
haem	O
(	O
binding	O
Kd	O
=	O
50	O
nmol	O
l	O
−	O
1	O
),	O
PGRMC1	O
forms	O
a	O
stable	O
dimer	O
(	O
dimerization	O
Kd	O
<<	O
3	O
.	O
5	O
μmol	O
l	O
−	O
1	O
)	O
through	O
stacking	O
of	O
the	O
two	O
open	O
surfaces	O
of	O
the	O
five	O
-	O
coordinated	O
haem	O
molecules	O
in	O
each	O
monomer	O
.	O
	O
While	O
proliferation	O
of	O
HCT116	O
cells	O
was	O
not	O
affected	O
by	O
knocking	O
down	O
PGRMC1	O
,	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
cells	O
were	O
more	O
sensitive	O
to	O
the	O
EGFR	O
inhibitor	O
erlotinib	O
than	O
control	O
HCT116	O
cells	O
,	O
and	O
the	O
knockdown	O
effect	O
was	O
reversed	O
by	O
co	O
-	O
expression	O
of	O
shRNA	O
-	O
resistant	O
wild	O
-	O
type	O
PGRMC1	O
but	O
not	O
of	O
the	O
Y113F	B-mutant
mutant	O
(	O
Fig	O
.	O
5b	O
).	O
	O
Furthermore	O
,	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
inhibited	O
spheroid	O
formation	O
of	O
HCT116	O
cells	O
in	O
culture	O
,	O
and	O
this	O
inhibition	O
was	O
reversed	O
by	O
co	O
-	O
expression	O
of	O
wild	O
-	O
type	O
PGRMC1	O
but	O
not	O
of	O
the	O
Y113F	B-mutant
mutant	O
(	O
Fig	O
.	O
5c	O
and	O
Supplementary	O
Fig	O
.	O
18	O
).	O
	O
The	O
Kd	O
value	O
of	O
PGRMC1	O
binding	O
to	O
CYP51	O
was	O
in	O
a	O
micromolar	O
range	O
and	O
comparable	O
with	O
those	O
of	O
other	O
haem	O
proteins	O
,	O
such	O
as	O
cytochrome	O
P450	O
reductase	O
and	O
neuroglobin	O
/	O
Gαi1	O
(	O
ref	O
.),	O
suggesting	O
that	O
haem	O
-	O
dependent	O
PGRMC1	O
interaction	O
with	O
CYP51	O
is	O
biologically	O
relevant	O
.	O
	O
In	O
this	O
study	O
,	O
we	O
showed	O
that	O
PGRMC1	O
dimerizes	O
by	O
stacking	O
interactions	O
of	O
haem	O
molecules	O
from	O
each	O
monomer	O
.	O
	O
In	O
the	O
current	O
study	O
,	O
the	O
Y113	O
residue	O
plays	O
a	O
crucial	O
role	O
for	O
the	O
haem	O
-	O
dependent	O
dimerization	O
of	O
PGRMC1	O
and	O
resultant	O
regulation	O
of	O
cancer	O
proliferation	O
and	O
chemoresistance	O
(	O
Figs	O
5c	O
and	O
6e	O
).	O
	O
Since	O
the	O
Y113	O
residue	O
is	O
involved	O
in	O
the	O
putative	O
consensus	O
motif	O
of	O
phosphorylation	O
by	O
tyrosine	O
kinases	O
such	O
as	O
Abl	O
and	O
Lck	O
,	O
we	O
investigated	O
whether	O
phosphorylated	O
Y113	O
is	O
present	O
in	O
HCT116	O
cells	O
by	O
ESI	O
-	O
MS	O
analysis	O
.	O
	O
We	O
showed	O
that	O
the	O
haem	O
-	O
mediated	O
dimer	O
of	O
PGRMC1	O
enables	O
interaction	O
with	O
different	O
subclasses	O
of	O
cytochromes	O
P450	O
(	O
CYP	O
)	O
(	O
Fig	O
.	O
6	O
).	O
	O
On	O
the	O
other	O
hand	O
,	O
Oda	O
et	O
al	O
.	O
reported	O
that	O
PGRMC1	O
had	O
no	O
effect	O
to	O
CYP2E1	O
and	O
CYP3A4	O
activities	O
in	O
HepG2	O
cell	O
.	O
	O
Besides	O
the	O
regulatory	O
roles	O
of	O
PGRMC1	O
/	O
Sigma	O
-	O
2	O
receptor	O
in	O
proliferation	O
and	O
chemoresistance	O
in	O
cancer	O
cells	O
(	O
ref	O
.),	O
recent	O
reports	O
show	O
that	O
PGRMC1	O
is	O
able	O
to	O
bind	O
to	O
amyloid	O
beta	O
oligomer	O
to	O
enhance	O
its	O
neurotoxicity	O
.	O
	O
Alzheimer	O
'	O
s	O
therapeutics	O
targeting	O
amyloid	O
beta	O
1	O
-	O
42	O
oligomers	O
II	O
:	O
Sigma	O
-	O
2	O
/	O
PGRMC1	O
receptors	O
mediate	O
Abeta	O
42	O
oligomer	O
binding	O
and	O
synaptotoxicity	O
	O
X	O
-	O
ray	O
crystal	O
structure	O
of	O
PGRMC1	O
.	O
	O
(	O
a	O
)	O
Structure	O
of	O
the	O
PGRMC1	O
dimer	O
formed	O
through	O
stacked	O
haems	O
.	O
	O
(	O
b	O
)	O
SV	O
-	O
AUC	O
analyses	O
of	O
the	O
wt	O
-	O
PGRMC1	O
and	O
the	O
C129S	B-mutant
mutant	O
(	O
a	O
.	O
a	O
.	O
44	O
–	O
195	O
)	O
in	O
the	O
presence	O
or	O
absence	O
of	O
haem	O
.	O
	O
(	O
f	O
)	O
HCT116	O
cells	O
expressing	O
control	O
shRNA	O
or	O
those	O
knocking	O
down	O
PGRMC1	O
(	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
)	O
were	O
treated	O
with	O
EGF	O
or	O
left	O
untreated	O
,	O
and	O
components	O
of	O
the	O
EGFR	O
signaling	O
pathway	O
were	O
detected	O
by	O
Western	O
blotting	O
.	O
	O
Stable	O
PGRMC1	B-mutant
-	I-mutant
knockdown	I-mutant
(	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
)	O
HCT116	O
cells	O
were	O
transiently	O
transfected	O
with	O
the	O
shRNA	O
-	O
resistant	O
expression	O
vector	O
of	O
wild	O
-	O
type	O
PGRMC1	O
(	O
wt	O
)	O
or	O
the	O
Y113F	B-mutant
mutant	O
(	O
Y113F	B-mutant
).	O
	O
(	O
b	O
)	O
Erlotinib	O
was	O
added	O
to	O
HCT116	O
(	O
control	O
)	O
cells	O
,	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
cells	O
or	O
PGRMC1	B-mutant
-	I-mutant
KD	I-mutant
cells	O
expressing	O
shRNA	O
-	O
resistant	O
PGRMC1	O
wt	O
or	O
Y113F	B-mutant
,	O
and	O
cell	O
viability	O
was	O
examined	O
by	O
MTT	O
assay	O
.	O
	O
The	O
graph	O
represents	O
mean	O
±	O
s	O
.	O
e	O
.	O
of	O
each	O
spheroid	O
size	O
.	O
*	O
P	O
<	O
0	O
.	O
01	O
using	O
ANOVA	O
with	O
Fischer	O
'	O
s	O
LSD	O
test	O
.	O
	O
Haem	O
-	O
dependent	O
PGRMC1	O
dimerization	O
enhances	O
tumour	O
chemoresistance	O
through	O
interaction	O
with	O
cytochromes	O
P450	O
.	O
	O
(	O
a	O
,	O
b	O
)	O
FLAG	O
-	O
PGRMC1	O
wild	O
-	O
type	O
(	O
wt	O
)	O
and	O
Y113F	B-mutant
mutant	O
proteins	O
(	O
a	O
.	O
a	O
.	O
44	O
–	O
195	O
),	O
in	O
either	O
apo	O
or	O
haem	O
-	O
bound	O
form	O
,	O
were	O
incubated	O
with	O
CYP1A2	O
(	O
a	O
)	O
or	O
CYP3A4	O
(	O
b	O
)	O
and	O
immunoprecipitated	O
with	O
anti	O
-	O
FLAG	O
antibody	O
-	O
conjugated	O
beads	O
.	O
	O
Schematic	O
diagram	O
for	O
the	O
regulation	O
of	O
PGRMC1	O
functions	O
.	O
	O
Differences	O
in	O
molecular	O
weights	O
of	O
the	O
wild	O
-	O
type	O
(	O
wt	O
;	O
a	O
)	O
and	O
the	O
C129S	B-mutant
mutant	O
(	O
b	O
)	O
PGRMC1	O
proteins	O
in	O
the	O
absence	O
(	O
apo	O
form	O
)	O
or	O
the	O
presence	O
of	O
haem	O
(	O
haem	O
-	O
bound	O
form	O
).	O
	O
Hotspot	O
autoimmune	O
T	O
cell	O
receptor	O
binding	O
underlies	O
pathogen	O
and	O
insulin	O
peptide	O
cross	O
-	O
reactivity	O
	O
Both	O
MHC	O
and	O
peptide	O
have	O
also	O
been	O
shown	O
to	O
undergo	O
structural	O
changes	O
upon	O
TCR	O
binding	O
,	O
mediating	O
an	O
induced	O
fit	O
between	O
the	O
TCR	O
and	O
pMHC	O
.	O
	O
We	O
recently	O
reported	O
that	O
the	O
1E6	O
human	O
CD8	O
+	O
T	O
cell	O
clone	O
—	O
which	O
mediates	O
the	O
destruction	O
of	O
β	O
cells	O
through	O
the	O
recognition	O
of	O
a	O
major	O
,	O
HLA	O
-	O
A	O
*	O
0201	O
–	O
restricted	O
,	O
preproinsulin	O
signal	O
peptide	O
(	O
ALWGPDPAAA15	O
–	O
24	O
)	O
—	O
can	O
recognize	O
upwards	O
of	O
1	O
million	O
different	O
peptides	O
.	O
	O
This	O
first	O
experimental	O
evidence	O
of	O
a	O
high	O
level	O
of	O
CD8	O
+	O
T	O
cell	O
cross	O
-	O
reactivity	O
in	O
a	O
human	O
autoimmune	O
disease	O
system	O
hinted	O
toward	O
molecular	O
mimicry	O
by	O
a	O
more	O
potent	O
pathogenic	O
peptide	O
as	O
a	O
potential	O
mechanism	O
leading	O
to	O
β	O
cell	O
destruction	O
.	O
	O
These	O
APLs	O
differed	O
from	O
the	O
natural	O
preproinsulin	O
peptide	O
by	O
up	O
to	O
7	O
of	O
10	O
residues	O
.	O
	O
Two	O
of	O
these	O
peptides	O
,	O
MVWGPDPLYV	O
and	O
RQFGPDWIVA	O
(	O
bold	O
text	O
signifies	O
amino	O
acids	O
that	O
are	O
different	O
from	O
the	O
index	O
preproinsulin	O
–	O
derived	O
sequence	O
),	O
are	O
contained	O
within	O
the	O
proteomes	O
of	O
the	O
human	O
pathogens	O
Bacteroides	O
fragilis	O
/	O
thetaiotaomicron	O
and	O
Clostridium	O
asparagiforme	O
,	O
respectively	O
.	O
	O
The	O
low	O
number	O
of	O
contacts	O
between	O
the	O
2	O
molecules	O
most	O
likely	O
contributed	O
to	O
the	O
weak	O
binding	O
affinity	O
of	O
the	O
interaction	O
.	O
	O
Although	O
the	O
1E6	O
TCR	O
formed	O
a	O
similar	O
overall	O
interaction	O
with	O
each	O
APL	O
,	O
the	O
stabilization	O
between	O
the	O
TCR	O
and	O
the	O
GPD	O
motif	O
enabled	O
fine	O
differences	O
in	O
the	O
contact	O
network	O
with	O
both	O
the	O
peptide	O
and	O
MHC	O
surface	O
that	O
allowed	O
discrimination	O
between	O
each	O
ligand	O
(	O
Figure	O
5	O
).	O
	O
These	O
data	O
demonstrated	O
that	O
the	O
unligated	O
structure	O
of	O
the	O
1E6	O
TCR	O
was	O
virtually	O
identical	O
to	O
its	O
ligated	O
counterparts	O
.	O
	O
Thus	O
,	O
we	O
performed	O
an	O
in	O
-	O
depth	O
thermodynamic	O
analysis	O
of	O
6	O
of	O
the	O
ligands	O
under	O
investigation	O
(	O
Figure	O
8	O
and	O
Supplemental	O
Table	O
3	O
).	O
	O
However	O
,	O
there	O
was	O
a	O
clear	O
switch	O
in	O
entropy	O
between	O
the	O
weaker	O
-	O
affinity	O
and	O
stronger	O
-	O
affinity	O
ligands	O
,	O
indicated	O
by	O
a	O
strong	O
Pearson	O
’	O
s	O
correlation	O
value	O
between	O
entropy	O
and	O
affinity	O
(	O
Pearson	O
’	O
s	O
correlation	O
value	O
0	O
.	O
93	O
,	O
P	O
=	O
0	O
.	O
007	O
).	O
	O
We	O
searched	O
a	O
database	O
of	O
over	O
1	O
,	O
924	O
,	O
572	O
unique	O
decamer	O
peptides	O
from	O
the	O
proteome	O
of	O
viral	O
pathogens	O
that	O
are	O
known	O
,	O
or	O
strongly	O
suspected	O
,	O
to	O
infect	O
humans	O
.	O
	O
This	O
notion	O
is	O
attractive	O
because	O
the	O
CDR	O
loops	O
,	O
which	O
form	O
the	O
TCR	O
antigen	O
-	O
binding	O
site	O
,	O
are	O
usually	O
the	O
most	O
flexible	O
part	O
of	O
the	O
TCR	O
and	O
have	O
the	O
ability	O
to	O
mold	O
around	O
differently	O
shaped	O
ligands	O
.	O
	O
This	O
motif	O
was	O
conserved	O
in	O
at	O
least	O
2	O
potential	O
foreign	O
peptides	O
,	O
originating	O
from	O
Herpes	O
simplex	O
virus	O
and	O
Pseudomonas	O
aeruginosa	O
,	O
enabling	O
TCR	O
recognition	O
of	O
foreign	O
epitopes	O
.	O
	O
We	O
have	O
previously	O
demonstrated	O
the	O
importance	O
of	O
the	O
GPD	O
motif	O
using	O
a	O
peptide	O
library	O
scan	O
,	O
as	O
well	O
as	O
a	O
CPL	O
scan	O
approach	O
.	O
	O
These	O
results	O
challenge	O
the	O
notion	O
that	O
the	O
most	O
potent	O
peptide	O
antigens	O
exhibit	O
the	O
greatest	O
pMHC	O
stability	O
and	O
have	O
implications	O
for	O
the	O
design	O
of	O
anchor	O
residue	O
–	O
modified	O
heteroclitic	O
peptides	O
for	O
vaccination	O
.	O
	O
These	O
parameters	O
aligned	O
well	O
with	O
structural	O
data	O
,	O
demonstrating	O
that	O
TCRs	O
engaged	O
pMHC	O
using	O
an	O
induced	O
fit	O
binding	O
mode	O
.	O
	O
These	O
differences	O
were	O
consistent	O
with	O
a	O
greater	O
degree	O
of	O
movement	O
between	O
the	O
unligated	O
and	O
ligated	O
pMHCs	O
for	O
the	O
weaker	O
ligands	O
,	O
suggesting	O
a	O
greater	O
requirement	O
for	O
disorder	O
-	O
to	O
-	O
order	O
changes	O
during	O
TCR	O
binding	O
.	O
	O
Indeed	O
,	O
we	O
found	O
over	O
50	O
decamer	O
peptides	O
from	O
the	O
proteome	O
of	O
likely	O
,	O
or	O
known	O
,	O
human	O
viral	O
pathogens	O
alone	O
that	O
contained	O
both	O
the	O
conserved	O
central	O
GPD	O
motif	O
and	O
anchor	O
residues	O
at	O
positions	O
2	O
and	O
10	O
that	O
would	O
enable	O
binding	O
to	O
HLA	O
-	O
A	O
*	O
02	O
:	O
01	O
.	O
	O
(	O
K	O
)	O
Effective	O
2D	O
affinity	O
plotted	O
against	O
1	O
/	O
EC50	O
showing	O
Pearson	O
’	O
s	O
coefficient	O
analysis	O
(	O
r	O
)	O
and	O
P	O
value	O
.	O
	O
(	O
A	O
)	O
Superposition	O
of	O
the	O
1E6	O
TCR	O
(	O
multicolored	O
illustration	O
)	O
in	O
complex	O
with	O
all	O
7	O
APLs	O
(	O
multicolored	O
sticks	O
)	O
and	O
the	O
A2	O
-	O
ALWGPDPAAA	O
ligand	O
using	O
the	O
HLA	O
-	O
A	O
*	O
0201	O
(	O
gray	O
illustration	O
)	O
molecule	O
to	O
align	O
all	O
of	O
the	O
structures	O
.	O
	O
The	O
MHCα1	O
helix	O
is	O
shown	O
in	O
gray	O
illustrations	O
.	O
	O
(	O
A	O
)	O
A2	O
-	O
MVWGPDPLYV	O
(	O
black	O
sticks	O
).	O
	O